Novel antigen-binding polypeptides and their uses

ABSTRACT

Novel antigen-binding polypeptides (ABP) and uses thereof are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 60/581,334, filed Jun. 18, 2004, U.S. provisional patentapplication Ser. No. 60/648,222, filed Jan. 28, 2005, and U.S.provisional patent application 60/654,018 filed Feb. 17, 2005, thespecifications of which are incorporated herein in their entirety.

FIELD OF THE INVENTION

This invention relates to novel antigen-binding polypeptides comprisingat least one non-naturally-encoded amino acid. The present inventionrelates generally to the field of the production and selection ofantigen-binding polypeptides by the methods of molecular biology, usingboth chemistry and recombinant DNA.

BACKGROUND OF THE INVENTION

A naturally produced antibody (Ab) is a tetrameric structure consistingof two identical immunoglobulin (Ig) heavy chains and two identicallight chains. The heavy and light chains of an Ab consist of differentdomains. Each light chain has one variable domain (VL) and one constantdomain (CL), while each heavy chain has one variable domain (VH) andthree or four constant domains (CH). Each domain, consisting of about110 amino acid residues, is folded into a characteristic β-sandwichstructure formed from two β-sheets packed against each other, theimmunoglobulin fold. The VL domains each have three complementaritydetermining regions (CDR1-3) and the VH domains each have up to fourcomplementarity determining regions (CDR1-4), that are loops, or turns,connecting β-strands at one end of the domains. The variable regions ofboth the light and heavy chains generally contribute to antigenspecificity, although the contribution of the individual chains tospecificity is not necessarily equal. Antibody molecules have evolved tobind to a large number of molecules by using randomized CDR loops.

Functional substructures of Abs can be prepared by proteolysis and byrecombinant methods. They include the Fab fragment, which comprises theVH-CH1 domains of the heavy chain and the VL-CL1 domains of the lightchain joined by a single interchain disulfide bond, and the Fv fragment,which comprises only the VH and VL domains, and the Fc portion whichcomprises the non-antigen binding region of the molecule. In some cases,a single VH domain retains significant affinity for antigen (Ward etal., 1989, Nature 341, 554-546). It has also been shown that a certainmonomeric κ light chain will specifically bind to its antigen. (L. Masatet al., 1994, PNAS 91:893-896). Separated light or heavy chains havesometimes been found to retain some antigen-binding activity as well(Ward et al., 1989, Nature 341, 554-546).

Another functional substructure is a single chain Fv (scFv), comprisedof the variable regions of the immunoglobulin heavy and light chain,covalently connected by a peptide linker (S-z Hu et al., 1996, CancerResearch, 56, 3055-3061). These small (Mr 25,000) proteins generallyretain specificity and affinity for antigen in a single polypeptide andcan provide a convenient building block for larger, antigen-specificmolecules. The short half-life of scFvs in the circulation limits theirtherapeutic utility in many cases.

A small protein scaffold called a “minibody” was designed using a partof the Ig VH domain as the template (Pessi et al., 1993, Nature 362,367-369). Minibodies with high affinity (dissociation constant (K_(d))about 10⁻⁷ M) to interleukin-6 were identified by randomizing loopscorresponding to CDR1 and CDR2 of VH and then selecting mutants usingthe phage display method (Martin et al., 1994, EMBO J. 13, 5303-5309).

Camels often lack variable light chain domains when IgG-like materialfrom their serum is analyzed, suggesting that sufficient antibodyspecificity and affinity can be derived from VH domains (three or fourCDR loops) alone. “Camelized” VH domains with high affinity have beenmade, and high specificity can be generated by randomizing only theCDR3.

An alternative to the “minibody” is the “diabody.” Diabodies are smallbivalent and bispecific antibody fragments, having two antigen-bindingsites. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) on the samepolypeptide chain (V_(H)-V_(L)). Diabodies are similar in size to theFab fragment. By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. These dimeric antibody fragments, or “diabodies,”are bivalent and bispecific. See, P. Holliger et al., PNAS 90:6444-6448(1993).

CDR peptides and organic CDR mimetics have been made (Dougall et al.,1994, Trends Biotechnol. 12, 372-379). CDR peptides are short, typicallycyclic, peptides which correspond to the amino acid sequences of CDRloops of antibodies. CDR loops are responsible for antibody-antigeninteractions. CDR peptides and organic CDR mimetics have been shown toretain some binding affinity (Smyth & von Itzstein, 1994, J. Am. Chem.Soc. 116, 2725-2733). Mouse CDRs have been grafted onto the human Igframework without the loss of affinity (Jones et al., 1986, Nature 321,522-525; Riechmann et al., 1988).

In the body, specific Abs are selected and amplified from a largelibrary (affinity maturation). The processes can be reproduced in vitrousing combinatorial library technologies. The successful display of Abfragments on the surface of bacteriophage has made it possible togenerate and screen a vast number of CDR mutations (McCafferty et al.,1990, Nature 348, 552-554; Barbas et al., 1991, Proc. Natl. Acad. Sci.USA 88, 7978-7982; Winter et al., 1994, Annu. Rev. Immunol. 12,433-455). An increasing number of Fabs and Fvs (and their derivatives)are produced by this technique. The combinatorial technique can becombined with Ab mimics.

A number of protein domains that could potentially serve as proteinscaffolds have been expressed as fusions with phage capsid proteins.Review in Clackson & Wells, Trends Biotechnol. 12:173-184 (1994).Several of these protein domains have already been used as scaffolds fordisplaying random peptide sequences, including bovine pancreatic trypsininhibitor (Roberts et al., PNAS 89:2429-2433 (1992)), human growthhormone (Lowman et al., Biochemistry 30:10832-10838 (1991)), Venturiniet al., Protein Peptide Letters 1:70-75 (1994)), and the IgG bindingdomain of Streptococcus (O'Neil et al., Techniques in Protein ChemistryV (Crabb, L., ed.) pp. 517-524, Academic Press, San Diego (1994)). Thesescaffolds have displayed a single randomized loop or region. Tendamistathas been used as a presentation scaffold on the filamentous phage M13(McConnell and Hoess, 1995, J. Mol. Biol. 250:460-470).

Receptor tyrosine kinases of the ErbB family play pivotal roles in cellgrowth and differentiation. Aberrant activation of these receptors isassociated with human cancers. Dimerization (the pairing of receptors)is essential to the signaling activity of all ErbB receptors. Blockingthe dimerization activity of ErbB2 has been shown to directly inhibitthe ability of ErbB2 to dimerize with other ErbB receptor proteins.Inhibiting receptor dimerization prevents the activation of ErbBsignaling pathways. An antagonistic molecule that down regulates ErbBsignaling could function as an anti-tumor agent. The ErbB signalingnetwork is currently a major target in the development of anti-tumordrugs. ErbB-1 is a specific receptor for EGF, while ErbB-2 has no knownnatural ligand. ErbB2 is able to form heterodimers with ErbB-1 uponaddition of EGF. ErbB2 also functions as the preferred dimerizationpartner for the kinase-dead ErbB-3 and for ErbB-4, which are bothreceptors for the neuregulins. The ErbB signaling network can also beactivated in an indirect manner during signaling by cytokines andligands of G-coupled protein receptors, indicating that it plays acentral role in the growth control of many different cell types.

The proto-oncogene c-erbB-1 encodes the epidermal growth factorreceptor. Its name originates from the viral homolog v-erbB which wasisolated from an avian erythroblastosis virus (AEV) where it wascontained as a fragment of the chicken c-ErbB-1 gene lacking theamino-terminal ligand-binding domain. Over expression of erbB-1 genesoccurs in a wide range of tumors, including squamous carcinomas ofvarious sites and adenocarcinomas. The human c-erbB-1 gene is located inthe chromosomal region 7p14 and 7p12.

The ErbB-2 proto-oncogene (also referred to as Neu, EGFR-2 or HER-2) isa member of the transmembrane receptor tyrosine kinase family, whichalso includes EGF receptor and EGFR-3 (HER-3 or ErbB-3). ErbB-2 encodesa transmembrane receptor-like glycoprotein of 185 kDa with intrinsictyrosine kinase activity. Although, ErbB-2 does not have any knownhigh-affinity ligands, its kinase activity can be activated withoutligand by either over expression or hetero-association with othermembers of the ErbB family of receptors. Amplification of the ErbB-2gene and over expression of its product has been detected in almost 40%of primary human breast tumors. ErbB-2 over expression is also observedin ovarian, gastric, salivary and non-small cell lung carcinomas. ErbB-2is activated by the neuregulins in heterodimers with the neuregulinreceptors ErbB-3 and ErbB-4. The humanized anti-ErbB-2 monoclonalantibody Herceptin (from monoclonal 4D5) has received FDA approval fortreatment of cancers that over express ErbB-2. Another anti-ErbB2antibody in development is Pertuzumab (from monoclonal 2C4). Specificinhibitors of the tyrosine kinase activity of ErbB-1 (EGF receptor) arealso in clinical trials.

Anti-ErbB2 antibodies are known in the art, and include but are notlimited to U.S. Pat. Nos. 4,753,894; 5,169,774; 5,677,171; 5,720,937;5,720,954; 5,725,856; 5,770,195; 5,772,997; 5,783,186; 6,054,561;6,165,464; 6,333,169; 6,015,567; 6,387,371; 6,399,063; 6,441,143;6,458,356; 6,627,196, each of which is incorporated by reference herein.

Covalent attachment of the hydrophilic polymer poly(ethylene glycol),abbreviated PEG, is a method of increasing water solubility,bioavailability, increasing serum half-life, increasing therapeutichalf-life, modulating immunogenicity, modulating biological activity, orextending the circulation time of many biologically active molecules,including proteins, peptides, and particularly hydrophobic molecules.PEG has been used extensively in pharmaceuticals, on artificialimplants, and in other applications where biocompatibility, lack oftoxicity, and lack of immunogenicity are of importance. In order tomaximize the desired properties of PEG, the total molecular weight andhydration state of the PEG polymer or polymers attached to thebiologically active molecule must be sufficiently high to impart theadvantageous characteristics typically associated with PEG polymerattachment, such as increased water solubility and circulating halflife, while not adversely impacting the bioactivity of the parentmolecule.

PEG derivatives are frequently linked to biologically active moleculesthrough reactive chemical functionalities, such as lysine, cysteine andhistidine residues, the N-terminus and carbohydrate moieties. Proteinsand other molecules often have a limited number of reactive sitesavailable for polymer attachment. Often, the sites most suitable formodification via polymer attachment play a significant role in receptorbinding, and are necessary for retention of the biological activity ofthe molecule. As a result, indiscriminate attachment of polymer chainsto such reactive sites on a biologically active molecule often leads toa significant reduction or even total loss of biological activity of thepolymer-modified molecule. R. Clark et al., (1996), J. Biol. Chem.,271:21969-21977. To form conjugates having sufficient polymer molecularweight for imparting the desired advantages to a target molecule, priorart approaches have typically involved random attachment of numerouspolymer arms to the molecule, thereby increasing the risk of a reductionor even total loss in bioactivity of the parent molecule.

Reactive sites that form the loci for attachment of PEG derivatives toproteins are dictated by the protein's structure. Proteins, includingenzymes, are composed of various sequences of alpha-amino acids, whichhave the general structure H₂N—CHR—COOH. The alpha amino moiety (H₂N—)of one amino acid joins to the carboxyl moiety (—COOH) of an adjacentamino acid to form amide linkages, which can be represented as—(NH—CHR—CO)_(n)—, where the subscript “n” can equal hundreds orthousands. The fragment represented by R can contain reactive sites forprotein biological activity and for attachment of PEG derivatives.

For example, in the case of the amino acid lysine, there exists an —NH₂moiety in the epsilon position as well as in the alpha position. Theepsilon —NH₂ is free for reaction under conditions of basic pH. Much ofthe art in the field of protein derivatization with PEG has beendirected to developing PEG derivatives for attachment to the epsilon—NH₂ moiety of lysine residues present in proteins. “Polyethylene Glycoland Derivatives for Advanced PEGylation”, Nektar Molecular EngineeringCatalog, 2003, pp. 1-17. These PEG derivatives all have the commonlimitation, however, that they cannot be installed selectively among theoften numerous lysine residues present on the surfaces of proteins. Thiscan be a significant limitation in instances where a lysine residue isimportant to protein activity, existing in an enzyme active site forexample, or in cases where a lysine residue plays a role in mediatingthe interaction of the protein with other biological molecules, as inthe case of receptor binding sites.

A second and equally important complication of existing methods forprotein PEGylation is that the PEG derivatives can undergo undesiredside reactions with residues other than those desired. Histidinecontains a reactive imino moiety, represented structurally as —N(H)—,but many chemically reactive species that react with epsilon —NH₂ canalso react with —N(H)—. Similarly, the side chain of the amino acidcysteine bears a free sulfhydryl group, represented structurally as —SH.In some instances, the PEG derivatives directed at the epsilon —NH₂group of lysine also react with cysteine, histidine or other residues.This can create complex, heterogeneous mixtures of PEG-derivatizedbioactive molecules and risks destroying the activity of the bioactivemolecule being targeted. It would be desirable to develop PEGderivatives that permit a chemical functional group to be introduced ata single site within the protein that would then enable the selectivecoupling of one or more PEG polymers to the bioactive molecule atspecific sites on the protein surface that are both well-defined andpredictable.

In addition to lysine residues, considerable effort in the art has beendirected toward the development of activated PEG reagents that targetother amino acid side chains, including cysteine, histidine and theN-terminus. See, e.g., U.S. Pat. No. 6,610,281 which is incorporated byreference herein, and “Polyethylene Glycol and Derivatives for AdvancedPEGylation”, Nektar Molecular Engineering Catalog, 2003, pp. 1-7. Acysteine residue can be introduced site-selectively into the structureof proteins using site-directed mutagenesis and other techniques knownin the art, and the resulting free sulfhydryl moiety can be reacted withPEG derivatives that bear thiol-reactive functional groups. Thisapproach is complicated, however, in that the introduction of a freesulfhydryl group can complicate the expression, folding and stability ofthe resulting protein. Thus, it would be desirable to have a means tointroduce a chemical functional group into bioactive molecules thatenables the selective coupling of one or more PEG polymers to theprotein while simultaneously being compatible with (i.e., not engagingin undesired side reactions with) sulfhydryls and other chemicalfunctional groups typically found in proteins.

As can be seen from a sampling of the art, many of these derivativesthat have been developed for attachment to the side chains of proteins,in particular, the —NH₂ moiety on the lysine amino acid side chain andthe —SH moiety on the cysteine side chain, have proven problematic intheir synthesis and use. Some form unstable linkages with the proteinthat are subject to hydrolysis and therefore decompose, degrade, or areotherwise unstable in aqueous environments, such as in the bloodstream.Some form more stable linkages, but are subject to hydrolysis before thelinkage is formed, which means that the reactive group on the PEGderivative may be inactivated before the protein can be attached. Someare somewhat toxic and are therefore less suitable for use in vivo. Someare too slow to react to be practically useful. Some result in a loss ofprotein activity by attaching to sites responsible for the protein'sactivity. Some are not specific in the sites to which they will attach,which can also result in a loss of desirable activity and in a lack ofreproducibility of results. In order to overcome the challengesassociated with modifying proteins with poly(ethylene glycol) moieties,PEG derivatives have been developed that are more stable (e.g., U.S.Pat. No. 6,602,498, which is incorporated by reference herein) or thatreact selectively with thiol moieties on molecules and surfaces (e.g.,U.S. Pat. No. 6,610,281, which is incorporated by reference herein).There is clearly a need in the art for PEG derivatives that arechemically inert in physiological environments until called upon toreact selectively to form stable chemical bonds.

Recently, an entirely new technology in the protein sciences has beenreported, which promises to overcome many of the limitations associatedwith site-specific modifications of proteins. Specifically, newcomponents have been added to the protein biosynthetic machinery of theprokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001),Science 292:498-500) and the eukaryote Sacchromyces cerevisiae (S.cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which hasenabled the incorporation of non-genetically encoded amino acids toproteins in vivo. A number of new amino acids with novel chemical,physical or biological properties, including photoaffinity labels andphotoisomerizable amino acids, keto amino acids, and glycosylated aminoacids have been incorporated efficiently and with high fidelity intoproteins in E. coli and in yeast in response to the amber codon, TAG,using this methodology. See, e.g., J. W. Chin et al., (2002), Journal ofthe American Chemical Society 124:9026-9027; J. W. Chin, & P. G.Schultz, (2002), Chem Bio Chem 11:1135-1137; J. W. Chin, et al., (2002),PNAS United States of America 99:11020-11024; and, L. Wang, & P. G.Schultz, (2002), Chem. Comm., 1-10. These studies have demonstrated thatit is possible to selectively and routinely introduce chemicalfunctional groups, such as ketone groups, alkyne groups and azidemoieties, that are not found in proteins, that are chemically inert toall of the functional groups found in the 20 common, genetically-encodedamino acids and that may be used to react efficiently and selectively toform stable covalent linkages.

The ability to incorporate non-genetically encoded amino acids intoproteins permits the introduction of chemical functional groups thatcould provide valuable alternatives to the naturally-occurringfunctional groups, such as the epsilon —NH₂ of lysine, the sulfhydryl—SH of cysteine, the imino group of histidine, etc. Certain chemicalfunctional groups are known to be inert to the functional groups foundin the 20 common, genetically-encoded amino acids but react cleanly andefficiently to form stable linkages. Azide and acetylene groups, forexample, are known in the art to undergo a Huisgen [3+2]cycloadditionreaction in aqueous conditions in the presence of a catalytic amount ofcopper. See, e.g., Tornoe, et al., (2002) Org. Chem. 67:3057-3064; and,Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. Byintroducing an azide moiety into a protein structure, for example, oneis able to incorporate a functional group that is chemically inert toamines, sulfhydryls, carboxylic acids, hydroxyl groups found inproteins, but that also reacts smoothly and efficiently with anacetylene moiety to form a cycloaddition product. Importantly, in theabsence of the acetylene moiety, the azide remains chemically inert andunreactive in the presence of other protein side chains and underphysiological conditions.

The present invention addresses, among other things, problems associatedwith the activity and production of antigen-binding polypeptides andfragments thereof, and also addresses the production of antigen-bindingpolypeptides with improved biological or pharmacological properties,such as improved therapeutic half-life.

BRIEF SUMMARY OF THE INVENTION

This invention provides antigen-binding polypeptides (ABP) comprisingone or more non-naturally encoded amino acids. In some embodiments, theABP comprises a complete antibody heavy chain. In some embodiments, theABP comprises a complete antibody light chain. In some embodiments, theABP comprises a variable region of an antibody light chain. In someembodiments, the ABP comprises a variable region of an antibody heavychain. In some embodiments, the ABP comprises at least one CDR of anantibody light chain. In some embodiments, the ABP comprises at leastone CDR of an antibody heavy chain. In some embodiments, the ABPcomprises at least one CDR of a light chain and at least one CDR of aheavy chain. In some embodiments, the ABP comprises a Fab. In someembodiments, the ABP comprises two or more Fab's. In some embodiments,the ABP comprises a scFv. In some embodiments, the ABP comprises two ormore scFv. In some embodiments, the ABP comprises a minibody. In someembodiments, the ABP comprises two or more minibodies. In someembodiments, the ABP comprises a diabody. In some embodiments, the ABPcomprises two or more diabodies. In some embodiments, the ABP comprisesa variable region of a light chain and a variable region of a heavychain. In some embodiments, the ABP comprises a complete light chain anda complete heavy chain. In some embodiments, the ABP comprises one ormore Fc domain or portion thereof. In some embodiments, the ABPcomprises a combination of any of the above embodiments. In someembodiments, the ABP comprises a homodimer, heterodimer, homomultimer orheteromultimer of any of the above embodiments. In some embodiments, theABP comprises a polypeptide that binds to a binding partner wherein thebinding partner comprises an antigen, a polypeptide, a nucleic acidmolecule, a polymer, or other molecule or substance. In someembodiments, the ABP is associated with a non-antibody scaffold moleculeor substance.

In some embodiments, the ABP comprises one or more post-translationalmodifications. In some embodiments, the ABP is linked to a linker,polymer, or biologically active molecule. In some embodiments, the ABPis linked to a bifunctional polymer, bifunctional linker, or at leastone additional ABP. In some embodiments, the ABP is linked to apolypeptide that is not an ABP. In some embodiments, the antigen-bindingpolypeptide comprising a non-naturally encoded amino acid is linked toone or more additional antigen-binding polypeptides which may alsocomprise a non-naturally encoded amino acid.

In some embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer. In some embodiments, the water soluble polymercomprises a poly(ethylene glycol) moiety. In some embodiments, thepoly(ethylene glycol) molecule is a bifunctional polymer. In someembodiments, the bifunctional polymer is linked to a second polypeptide.In some embodiments, the second polypeptide is an antigen-bindingpolypeptide.

In some embodiments, the antigen-binding polypeptide comprises at leasttwo amino acids linked to a water soluble polymer comprising apoly(ethylene glycol) moiety. In some embodiments, at least one aminoacid is a non-naturally encoded amino acid.

In some embodiments, the antigen-binding polypeptide comprises asubstitution, addition or deletion that modulates affinity of theantigen-binding polypeptide for an antigen when compared with theaffinity of the corresponding antigen-binding polypeptide without thesubstitution, addition or deletion. In some embodiments, theantigen-binding polypeptide comprises a substitution, addition, ordeletion that increases the stability of the antigen-binding polypeptidewhen compared with the stability of the corresponding antigen-bindingpolypeptide without the substitution, addition or deletion. In someembodiments, the antigen-binding polypeptide comprises a substitution,addition, or deletion that modulates the immunogenicity of theantigen-binding polypeptide when compared with the immunogenicity of thecorresponding antigen-binding polypeptide without the substitution,addition or deletion. In some embodiments, the antigen-bindingpolypeptide comprises a substitution, addition, or deletion thatmodulates serum half-life or circulation time of the antigen-bindingpolypeptide when compared with the serum half-life or circulation timeof the corresponding antigen-binding polypeptide without thesubstitution, addition or deletion.

In some embodiments, the antigen-binding polypeptide comprises asubstitution, addition, or deletion that increases the aqueoussolubility of the corresponding antigen-binding polypeptide whencompared to the aqueous solubility of the corresponding antigen-bindingpolypeptide without the substitution, addition, or deletion. In someembodiments, the antigen-binding polypeptide comprises a substitution,addition, or deletion that increases the solubility of theantigen-binding polypeptide produced in a host cell when compared to thesolubility of the corresponding antigen-binding polypeptide without thesubstitution, addition, or deletion. In some embodiments, theantigen-binding polypeptide comprises a substitution, addition, ordeletion that increases the expression of the antigen-bindingpolypeptide in a host cell or synthesized in vitro when compared to theexpression of the corresponding antigen-binding polypeptide without thesubstitution, addition, or deletion. In some embodiments, theantigen-binding polypeptide comprises a substitution, addition, ordeletion that increases protease resistance of the antigen-bindingpolypeptide when compared to protease resistance of the correspondingantigen-binding polypeptide without the substitution, addition, ordeletion.

In some embodiments the amino acid substitutions in the ABP may be withnaturally occurring or non-naturally occurring amino acids, providedthat at least one substitution is with a non-naturally encoded aminoacid.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group, an acetyl group, an aminooxy group, a hydrazine group, ahydrazide group, a semicarbazide group, an azide group, or an alkynegroup.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group. In some embodiments, the non-naturally encoded aminoacid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, an alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group.

In some embodiments, the non-naturally encoded amino acid comprises anaminooxy group. In some embodiments, the non-naturally encoded aminoacid comprises a hydrazide group. In some embodiments, the non-naturallyencoded amino acid comprises a hydrazine group. In some embodiments, thenon-naturally encoded amino acid residue comprises a semicarbazidegroup.

In some embodiments, the non-naturally encoded amino acid residuecomprises an azide group. In some embodiments, the non-naturally encodedamino acid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the non-naturally encoded amino acid comprises analkyne group. In some embodiments, the non-naturally encoded amino acidhas the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; X is O, N, S or not present; m is 0-10, R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the polypeptide is an agonist, partial agonist,antagonist, partial antagonist, or inverse agonist of at least oneactivity of the antigen. In some embodiments, the agonist, partialagonist, antagonist, partial antagonist, or inverse agonist comprises anon-naturally encoded amino acid linked to a water soluble polymer. Insome embodiments, the water soluble polymer comprises a poly(ethyleneglycol) moiety. In some embodiments, the agonist, partial agonist,antagonist, partial antagonist, or inverse agonist comprises anon-naturally encoded amino acid and one or more post-translationalmodification, linker, polymer, or biologically active molecule.

The present invention also provides isolated nucleic acids comprising apolynucleotide that encodes an antigen-binding polypeptide wherein thepolynucleotide comprises at least one selector codon including, but notlimited to, SEQ ID NO: 18, 20, 22, 25, 27, 29. In some embodiments, theselector codon is selected from the group consisting of an amber codon,ochre codon, opal codon, a unique codon, a rare codon, and a four-basecodon.

The present invention also provides methods of making an antigen-bindingpolypeptide linked to a water soluble polymer. In some embodiments, themethod comprises contacting an isolated antigen-binding polypeptidecomprising a non-naturally encoded amino acid with a water solublepolymer comprising a moiety that reacts with the non-naturally encodedamino acid. In some embodiments, the non-naturally encoded amino acidincorporated into the antigen-binding polypeptide is reactive toward awater soluble polymer that is otherwise unreactive toward any of the 20common amino acids. In some embodiments, the non-naturally encoded aminoacid incorporated into the antigen-binding polypeptide is reactivetoward a linker, polymer, or biologically active molecule that isotherwise unreactive toward any of the 20 common amino acids.

In some embodiments, the antigen-binding polypeptide linked to the watersoluble polymer is made by reacting an antigen-binding polypeptidecomprising a carbonyl-containing amino acid with a poly(ethylene glycol)molecule comprising an aminooxy, hydrazine, hydrazide or semicarbazidegroup. In some embodiments, the aminooxy, hydrazine, hydrazide orsemicarbazide group is linked to the poly(ethylene glycol) moleculethrough an amide linkage.

In some embodiments, the antigen-binding polypeptide linked to the watersoluble polymer is made by reacting a poly(ethylene glycol) moleculecomprising a carbonyl group with a polypeptide comprising anon-naturally encoded amino acid that comprises an aminooxy, hydrazine,hydrazide or semicarbazide group.

In some embodiments, the antigen-binding polypeptide linked to the watersoluble polymer is made by reacting an antigen-binding polypeptidecomprising an alkyne-containing amino acid with a poly(ethylene glycol)molecule comprising an azide moiety. In some embodiments, the azide oralkyne group is linked to the poly(ethylene glycol) molecule through anamide linkage.

In some embodiments, the antigen-binding polypeptide linked to the watersoluble polymer is made by reacting an antigen-binding polypeptidecomprising an azide-containing amino acid with a poly(ethylene glycol)molecule comprising an alkyne moiety. In some embodiments, the azide oralkyne group is linked to the poly(ethylene glycol) molecule through anamide linkage.

In some embodiments, the poly(ethylene glycol) molecule has a molecularweight of between about 0.1 kDa and about 100 kDa. In some embodiments,the poly(ethylene glycol) molecule has a molecular weight of between 0.1kDa and 50 kDa.

In some embodiments, the poly(ethylene glycol) molecule is a branchedpolymer. In some embodiments, each branch of the poly(ethylene glycol)branched polymer has a molecular weight of between 1 kDa and 100 kDa, orbetween 1 kDa and 50 kDa.

In some embodiments, the water soluble polymer linked to theantigen-binding polypeptide comprises a polyalkylene glycol moiety. Insome embodiments, the non-naturally encoded amino acid residueincorporated into the antigen-binding polypeptide comprises a carbonylgroup, an aminooxy group, a hydrazide group, a hydrazine, asemicarbazide group, an azide group, or an alkyne group. In someembodiments, the non-naturally encoded amino acid residue incorporatedinto the ABP comprises a carbonyl moiety and the water soluble polymercomprises an aminooxy, hydrazide, hydrazine, or semicarbazide moiety. Insome embodiments, the non-naturally encoded amino acid residueincorporated into the antigen-binding polypeptide comprises an alkynemoiety and the water soluble polymer comprises an azide moiety. In someembodiments, the non-naturally encoded amino acid residue incorporatedinto the antigen-binding polypeptide comprises an azide moiety and thewater soluble polymer comprises an alkyne moiety.

The present invention also provides compositions comprising anantigen-binding polypeptide comprising a non-naturally-encoded aminoacid and a pharmaceutically acceptable carrier. In some embodiments, thenon-naturally encoded amino acid is linked to a water soluble polymer.

The present invention also provides cells comprising a polynucleotideencoding the antigen-binding polypeptide comprising a selector codon. Insome embodiments, the cells comprise an orthogonal RNA synthetase and/oran orthogonal tRNA for substituting a non-naturally encoded amino acidinto the antigen-binding polypeptide.

The present invention also provides methods of making an antigen-bindingpolypeptide comprising a non-naturally encoded amino acid. In someembodiments, the methods comprise culturing cells comprising apolynucleotide or polynucleotides encoding an antigen-bindingpolypeptide, an orthogonal RNA synthetase and/or an orthogonal tRNAunder conditions to permit expression of the antigen-bindingpolypeptide; and purifying the antigen-binding polypeptide from thecells and/or culture medium.

The present invention also provides methods of increasing therapeutichalf-life, serum half-life or circulation time of the antigen-bindingpolypeptides. The present invention also provides methods of modulatingimmunogenicity of the antigen-binding polypeptides. In some embodiments,the methods comprise substituting a non-naturally encoded amino acid forany one or more amino acids in naturally occurring antigen-bindingpolypeptides and/or linking the antigen-binding polypeptide to a linker,a polymer, a water soluble polymer, or a biologically active molecule.

The present invention also provides methods of treating a patient inneed of such treatment with an effective amount of an antigen-bindingpolypeptide of the present invention. In some embodiments, the methodscomprise administering to the patient a therapeutically-effective amountof a pharmaceutical composition comprising an antigen-bindingpolypeptide comprising a non-naturally-encoded amino acid and apharmaceutically acceptable carrier. In some embodiments, thenon-naturally encoded amino acid is linked to a water soluble polymer.

The present invention also provides antigen-binding polypeptidescomprising a sequence shown in SEQ ID NO: 19, 21, 23, 24, 26, 28, 30, 31and fragments thereof, or any other antigen-binding polypeptidesequence, except that at least one amino acid is substituted by anon-naturally encoded amino acid. In some embodiments, the non-naturallyencoded amino acid is linked to a water soluble polymer. In someembodiments, the water soluble polymer comprises a poly(ethylene glycol)moiety. In some embodiments, the non-naturally encoded amino acidcomprises a carbonyl group, an aminooxy group, a hydrazide group, ahydrazine group, a semicarbazide group, an azide group, or an alkynegroup.

The present invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and an antigen-bindingpolypeptide comprising the sequence shown in SEQ ID NO: 19, 21, 23, 24,26, 28, 30, 31 and fragments thereof, or any other antigen-bindingpolypeptide sequence, wherein at least one amino acid is substituted bya non-naturally encoded amino acid. In some embodiments, thenon-naturally encoded amino acid comprises a saccharide moiety. In someembodiments, the water soluble polymer is linked to the polypeptide viaa saccharide moiety. In some embodiments, a linker, polymer, orbiologically active molecule is linked to the antigen-bindingpolypeptide via a saccharide moiety.

The present invention also provides an antigen-binding polypeptidecomprising a water soluble polymer linked by a covalent bond to theantigen-binding polypeptide at a single amino acid. In some embodiments,the water soluble polymer comprises a poly(ethylene glycol) moiety. Insome embodiments, the amino acid covalently linked to the water solublepolymer is a non-naturally encoded amino acid present in thepolypeptide.

The present invention provides an antigen-binding polypeptide comprisingat least one linker, polymer, or biologically active molecule, whereinsaid linker, polymer, or biologically active molecule is attached to thepolypeptide through a functional group of a non-naturally encoded aminoacid ribosomally incorporated into the polypeptide. In some embodiments,the polypeptide is monoPEGylated. The present invention also provides aABP polypeptide comprising a linker, polymer, or biologically activemolecule that is attached to one or more non-naturally encoded aminoacid wherein said non-naturally encoded amino acid is ribosomallyincorporated into the polypeptide at pre-selected sites.

In another embodiment, conjugation of the antigen-binding polypeptidecomprising one or more non-naturally occurring amino acids to anothermolecule, including but not limited to PEG, provides substantiallypurified antigen-binding polypeptide due to the unique chemical reactionutilized for conjugation to the non-natural amino acid. Conjugation ofthe antigen-binding polypeptide comprising one or more non-naturallyencoded amino acids to another molecule, such as PEG, may be performedwith other purification techniques performed prior to or following theconjugation step to provide substantially pure antigen-bindingpolypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—A diagram of the general structure of an antibody molecule (IgG)and its antigen-binding portions is shown. The CDR's are containedwithin the antigen recognition site.

FIG. 2—Constructs used for periplasmic (FIG. 2, Panel A) and cytoplasmic(FIG. 2, Panel B) expression/suppression of scFv-108 are shown.Locations of the amber stop codons are indicated. Bicistronic cassetteused for expression/suppression of the Fab-108 fragment (FIG. 2, PanelC) is shown. Constructs used for periplasmic expression/suppression ofscFv-4D5 fragments are shown (FIG. 2, Panel D and Panel E). A cistronfor expression/suppression of Fab-4D5 fragment is shown (FIG. 2, PanelF).

FIG. 3—Suppression (FIG. 3, Panel A) of amber mutations in the secondserine of the GlySer linker (S131Am) and analysis of IMAC purificationof the corresponding pAcF-containing scFv (FIG. 3, Panel B) are shown.

FIG. 4—Suppression of an amber mutation in the VL chain (L156) duringcytoplasmic expression of a scFv is shown.

FIG. 5—PEGylation and dimerization of pAcF-scFv-108 fragments is shownin FIG. 5, Panel A. Position of the mono-PEGylated scFv and the dimerare indicated by the single and double arrowheads respectively. FIG. 5,Panel B shows PEGylation of pAcF-scFv-108 fragment-(S136). FIG. 5, PanelC shows that no PEGylation of WT scFv fragments was observed.

FIG. 6—A gel showing fractions taken during purification of scFv-108homodimers is shown.

FIG. 7—Binding of pAcF or pAcF-PEG-containing scFv proteins to A431cells expressing EGF receptors are shown in FIG. 7, Panels A-C.

FIG. 8—A gel showing pAcF and pAcF-PEG-containing Fab fragments of mAb108 is shown as FIG. 8, Panel A. Binding of Fab fragments of mAb 108 toA431 cells expressing EGF receptors are shown in FIG. 8, Panels B-D.

FIG. 9—An example of a hetero-bifunctional ABP of the present inventionis shown.

FIG. 10—Gels showing the suppression of an amber mutation in the secondserine of the GlySer linker of the C-terminal (FIG. 10, Panel A) orN-terminal scFv-4D5 (FIG. 10, Panel B) fragments are shown.

FIG. 11—SDS-PAGE analysis is shown of pAcF-Fab-4D5-(K139) andFab-4D5-cys under both reducing and non-reducing conditions (FIG. 11,Panel A). FIG. 11, Panel B shows a Western blot of the samples shown inFIG. 11, Panel A with an anti-His antibody.

FIG. 12—HIV-1 neutralizing human Fab 4E10 linked to peptide T20 isshown.

FIG. 13—A diagram of a dimerization procedure is shown.

FIG. 14—Non-reducing (FIG. 14, Panel A) and reducing (FIG. 14, Panel B)SDS-PAGE analysis of scFv dimer formation is shown.

FIG. 15—SDS-PAGE analysis of purified scFv dimer is shown.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to an “antigen-bindingpolypeptide” or “ABP” is a reference to one or more such proteins andincludes equivalents thereof known to those skilled in the art, and soforth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

The term “substantially purified” refers to an ABP that may besubstantially or essentially free of components that normally accompanyor interact with the protein as found in its naturally occurringenvironment, i.e. a native cell, or host cell in the case ofrecombinantly produced ABP. ABP that may be substantially free ofcellular material includes preparations of protein having less thanabout 30%, less than about 25%, less than about 20%, less than about15%, less than about 10%, less than about 5%, less than about 4%, lessthan about 3%, less than about 2%, or less than about 1% (by dry weight)of contaminating protein. When the ABP or variant thereof isrecombinantly produced by the host cells, the protein may be present atabout 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about4%, about 3%, about 2%, or about 1% or less of the dry weight of thecells. When the ABP or variant thereof is recombinantly produced by thehost cells, the protein may be present in the culture medium at about 5g/L, about 4 g/L, about 3 g/L, about 2 g/L, about 1 g/L, about 750 mg/L,about 500 mg/L, about 250 mg/L, about 100 mg/L, about 50 mg/L, about 10mg/L, or about 1 mg/L or less of the dry weight of the cells. Thus,“substantially purified” ABP as produced by the methods of the presentinvention may have a purity level of at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, specifically, a purity level of at least about 75%, 80%, 85%, andmore specifically, a purity level of at least about 90%, a purity levelof at least about 95%, a purity level of at least about 99% or greateras determined by appropriate methods such as SDS/PAGE analysis, RP-HPLC,SEC, and capillary electrophoresis.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

As used herein, the term “medium” or “media” includes any culturemedium, solution, solid, semi-solid, or rigid support that may supportor contain any host cell, including bacterial host cells, yeast hostcells, insect host cells, plant host cells, eukaryotic host cells,mammalian host cells, CHO cells or E. coli, and cell contents. Thus, theterm may encompass medium in which the host cell has been grown, e.g.,medium into which the ABP has been secreted, including medium eitherbefore or after a proliferation step. The term also may encompassbuffers or reagents that contain host cell lysates, such as in the casewhere the ABP is produced intracellularly and the host cells are lysedor disrupted to release the ABP.

“Reducing agent,” as used herein with respect to protein refolding, isdefined as any compound or material which maintains sulfhydryl groups inthe reduced state and reduces intra- or intermolecular disulfide bonds.Suitable reducing agents include, but are not limited to, dithiothreitol(DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine(2-aminoethanethiol), and reduced glutathione. It is readily apparent tothose of ordinary skill in the art that a wide variety of reducingagents are suitable for use in the methods and compositions of thepresent invention.

“Oxidizing agent,” as used hereinwith respect to protein refolding, isdefined as any compound or material which is capable of removing anelectron from a compound being oxidized. Suitable oxidizing agentsinclude, but are not limited to, oxidized glutathione, cystine,cystamine, oxidized dithiothreitol, oxidized erythritol, and oxygen. Itis readily apparent to those of ordinary skill in the art that a widevariety of oxidizing agents are suitable for use in the methods of thepresent invention.

“Denaturing agent” or “denaturant,” as used herein, is defined as anycompound or material which will cause a reversible unfolding of aprotein. The strength of a denaturing agent or denaturant will bedetermined both by the properties and the concentration of theparticular denaturing agent or denaturant. Suitable denaturing agents ordenaturants may be chaotropes, detergents, organic solvents, watermiscible solvents, phospholipids, or a combination of two or more suchagents. Suitable chaotropes include, but are not limited to, urea,guanidine, and sodium thiocyanate. Useful detergents may include, butare not limited to, strong detergents such as sodium dodecyl sulfate, orpolyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mildnon-ionic detergents (e.g., digitonin), mild cationic detergents such asN->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents(e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergentsincluding, but not limited to, sulfobetaines (Zwittergent),3-(3-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate(CHAPSO). Organic, water miscible solvents such as acetonitrile, loweralkanols (especially C₂-C₄ alkanols such as ethanol or isopropanol), orlower alkandiols (especially C₂-C₄ alkandiols such as ethylene-glycol)may be used as denaturants. Phospholipids useful in the presentinvention may be naturally occurring phospholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, andphosphatidylinositol or synthetic phospholipid derivatives or variantssuch as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms disulfide bond containing polypeptides from animproperly folded or unfolded state to a native or properly foldedconformation with respect to disulfide bonds.

“Cofolding,” as used herein, refers specifically to refolding processes,reactions, or methods which employ at least two polypeptides whichinteract with each other and result in the transformation of unfolded orimproperly folded polypeptides to native, properly folded polypeptides.

Antibodies are proteins, which exhibit binding specificity to a specificantigen. Native antibodies are usually heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two identical light (L) chains andtwo identical heavy (H) chains. Each light chain is linked to a heavychain by one covalent disulfide bond, while the number of disulfidelinkages varies between the heavy chains of different immunoglobulinisotypes. Each heavy and light chain also has regularly spacedintrachain disulfide bridges. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called Complementarity DeterminingRegions (CDRs) both in the light chain and the heavy chain variabledomains. The more highly conserved portions of the variable domains arecalled the framework regions (FR). The variable domains of native heavyand light chains each comprise four FR regions, largely adopting aβ-sheet configuration, connected by three or four CDRs, which form loopsconnecting, and in some cases forming part of, the β-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).

The constant domains are not involved directly in binding an antibody toan antigen, but exhibit various effector functions. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavychain constant regions that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ and μ, respectively. Of thevarious human immunoglobulin classes, only human IgG1, IgG2, IgG3 andIgM are known to activate complement.

In vivo, affinity maturation of antibodies is driven by antigenselection of higher affinity antibody variants which are made primarilyby somatic hypermutagenesis. A “repertoire shift” also often occurs inwhich the predominant germline genes of the secondary or tertiaryresponse are seen to differ from those of the primary or secondaryresponse.

The affinity maturation process of the immune system may be replicatedby introducing mutations into antibody genes in vitro and using affinityselection to isolate mutants with improved affinity. Such mutantantibodies can be displayed on the surface of filamentous bacteriophageor microorganisms such as yeast, and antibodies can be selected by theiraffinity for antigen or by their kinetics of dissociation (off-rate)from antigen. Hawkins et al. J. Mol. Biol. 226:889-896 (1992). CDRwalking mutagenesis has been employed to affinity mature humanantibodies which bind the human envelope glycoprotein gp120 of humanimmunodeficiency virus type 1 (HIV-1) (Barbas III et al. PNAS (USA) 91:3809-3813 (1994); and Yang et al. J. Mol. Biol. 254:392-403 (1995)); andan anti-c-erbB-2 single chain Fv fragment (Schier et al. J. Mol. Biol.263:551567 (1996)). Antibody chain shuffling and CDR mutagenesis wereused to affinity mature a high-affinity human antibody directed againstthe third hypervariable loop of HIV (Thompson et al. J. Mol. Biol.256:77-88 (1996)). Balint and Larrick Gene 137:109-118 (1993) describe acomputer-assisted oligodeoxyribonucleotide-directed scanning mutagenesiswhereby all CDRs of a variable region gene are simultaneously andthoroughly searched for improved variants. An αvβ3-specific humanizedantibody was affinity matured using an initial limited mutagenesisstrategy in which every position of all six CDRs was mutated followed bythe expression and screening of a combinatorial library including thehighest affinity mutants (Wu et al. PNAS (USA) 95: 6037-6-42 (1998)).Phage displayed antibodies are reviewed in Chiswell and McCaffertyTIBTECH 10:80-84 (1992); and Rader and Barbas III Current Opinion inBiotech. 8:503-508 (1997). In each case where mutant antibodies withimproved affinity compared to a parent antibody are reported in theabove references, the mutant antibody has amino acid substitutions in aCDR.

By “affinity maturation” herein is meant the process of enhancing theaffinity of an antibody for its antigen. Methods for affinity maturationinclude but are not limited to computational screening methods andexperimental methods.

By “antibody” herein is meant a protein consisting of one or morepolypeptides substantially encoded by all or part of the antibody genes.The immunoglobulin genes include, but are not limited to, the kappa,lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon and muconstant region genes, as well as the myriad immunoglobulin variableregion genes. Antibody herein is meant to include full-length antibodiesand antibody fragments, and include antibodies that exist naturally inany organism or are engineered (e.g. are variants).

By “antibody fragment” is meant any form of an antibody other than thefull-length form. Antibody fragments herein include antibodies that aresmaller components that exist within full-length antibodies, andantibodies that have been engineered. Antibody fragments include but arenot limited to Fv, Fc, Fab, and (Fab′)₂, single chain Fv (scFv),diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies,CDR1, CDR2, CDR3, combinations of CDR's, variable regions, frameworkregions, constant regions, and the like (Maynard & Georgiou, 2000, Annu.Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol.9:395-402).

By “computational screening method” herein is meant any method fordesigning one or more mutations in a protein, wherein said methodutilizes a computer to evaluate the energies of the interactions ofpotential amino acid side chain substitutions with each other and/orwith the rest of the protein.

By “Fc” herein is meant the portions of an antibody that are comprisedof immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3). Fc may also includeany residues which exist in the N-terminal hinge between Cγ2 and Cγ1(Cγ1). Fc may refer to this region in isolation, or this region in thecontext of an antibody or antibody fragment. Fc also includes anymodified forms of Fc, including but not limited to the native monomer,the native dimer (disulfide bond linked), modified dimers (disulfideand/or non-covalently linked), and modified monomers (i.e.,derivatives).

By “full-length antibody” herein is meant the structure that constitutesthe natural biological form of an antibody H and/or L chain. In mostmammals, including humans and mice, this form is a tetramer and consistsof two identical pairs of two immunoglobulin chains, each pair havingone light and one heavy chain, each light chain comprisingimmunoglobulin domains V_(L) and C_(L), and each heavy chain comprisingimmunoglobulin domains V_(H), Cγ1, Cγ2, and Cγ3. In each pair, the lightand heavy chain variable regions (V_(L) and V_(H)) are togetherresponsible for binding to an antigen, and the constant regions (C_(L),Cγ1, Cγ2, and Cγ3, particularly Cγ2, and Cγ3) are responsible forantibody effector functions. In some mammals, for example in camels andllamas, full-length antibodies may consist of only two heavy chains,each heavy chain comprising immunoglobulin domains V_(H), Cγ2, and Cγ3.

By “immunoglobulin (Ig)” herein is meant a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes.Immunoglobulins include but are not limited to antibodies.Immunoglobulins may have a number of structural forms, including but notlimited to full-length antibodies, antibody fragments, and individualimmunoglobulin domains including but not limited to V_(H), Cγ1, Cγ2,Cγ3, V_(L), and C_(L).

By “immunoglobulin (Ig) domain” herein is meant a protein domainconsisting of a polypeptide substantially encoded by an immunoglobulingene. Ig domains include but are not limited to V_(H), Cγ1, Cγ2, Cγ3,V_(L), and C_(L) as is shown in FIG. 1.

By “variant protein sequence” as used herein is meant a protein sequencethat has one or more residues that differ in amino acid identity fromanother similar protein sequence. Said similar protein sequence may bethe natural wild type protein sequence, or another variant of the wildtype sequence. In general, a starting sequence is referred to as a“parent” sequence, and may either be a wild type or variant sequence.For example, preferred embodiments of the present invention may utilizehumanized parent sequences upon which computational analyses are done tomake variants.

By “variable region” of an antibody herein is meant a polypeptide orpolypeptides composed of the V_(H) immunoglobulin domain, the V_(L)immunoglobulin domains, or the V_(H) and V_(L) immunoglobulin domains asis shown in FIG. 1 (including variants). Variable region may refer tothis or these polypeptides in isolation, as an Fv fragment, as a scFvfragment, as this region in the context of a larger antibody fragment,or as this region in the context of a full-length antibody or analternative, non-antibody scaffold molecule.

The present invention may be applied to antibodies obtained from a widerange of sources. The antibody may be substantially encoded by anantibody gene or antibody genes from any organism, including but notlimited to humans, mice, rats, rabbits, camels, llamas, dromedaries,monkeys, particularly mammals and particularly human and particularlymice and rats. In one embodiment, the antibody may be fully human,obtained for example from a patient or subject, by using transgenic miceor other animals (Bruggemann & Taussig, 1997, Curr. Opin. Biotechnol.8:455-458) or human antibody libraries coupled with selection methods(Griffiths & Duncan, 1998, Curr. Opin. Biotechnol. 9:102-108). Theantibody may be from any source, including artificial or naturallyoccurring. For example the present invention may utilize an engineeredantibody, including but not limited to chimeric antibodies and humanizedantibodies (Clark, 2000, Immunol. Today 21:397-402) or derived from acombinatorial library. In addition, the antibody being optimized may bean engineered variant of an antibody that is substantially encoded byone or more natural antibody genes. For example, in one embodiment theantibody being optimized is an antibody that has been identified byaffinity maturation.

With respect to ABP's of the invention, the term “antigenicallyspecific” or “specifically binds” refers to ABP's that bind to one ormore epitopes of an antigen or binding partner of interest, but which donot substantially recognize and bind other molecules in a samplecontaining a mixed population of antigens.

The term “bispecific ABP” or “multispecific ABP” as used herein refersto an ABP comprising two or more antigen-binding sites or bindingpartner binding sites, a first binding site having affinity for a firstantigen or epitope and a second binding site having binding affinity fora second antigen or epitope distinct from the first.

The term “epitope” as used herein refers to a site on an antigen orbinding partner that is recognized by an ABP. An epitope may be a linearor conformationally formed sequence or shape of amino acids, if theantigen comprises a polypeptide. An epitope may also be any location onany type of antigen where an ABP binds to the antigen.

As used herein, “antigen-binding polypeptide” or “ABP” shall includethose polypeptides and proteins that have at least the biologicalactivity of specific binding to a particular binding partner such asantigen, as well as ABP analogs, ABP isoforms, ABP mimetics, ABPfragments, hybrid ABP proteins, fusion proteins, oligomers andmultimers, homologues, glycosylation pattern variants, and muteins,thereof, regardless of the biological activity of same, and furtherregardless of the method of synthesis or manufacture thereof including,but not limited to, recombinant (whether produced from cDNA, genomicDNA, synthetic DNA or other form of nucleic acid), in vitro, in vivo, bymicroinjection of nucleic acid molecules, synthetic, transgenic, andgene activated methods. Specific examples of ABP include, but are notlimited to, antibody molecules, heavy chain, light chain, variableregion, CDR, Fab, scFv, alternative scaffold non-antibody molecules,ligands, receptors, peptides, or any amino acid sequence that binds toan antigen.

The term “ABP” or “antigen-binding polypeptide” refers to an ABP asdescribed above, as well as a polypeptide that retains at least onebiological activity of a naturally-occurring antibody, including but notlimited to, activities other than antigen binding. Activities other thanantigen binding include, but are not limited to, any one or more of theactivities associated with the Fc.

Antigen-binding polypeptides include the pharmaceutically acceptablesalts and prodrugs, and prodrugs of the salts, polymorphs, hydrates,solvates, biologically-active fragments, biologically-active variantsand stereoisomers of the naturally-occurring human ABP as well asagonist, mimetic, and antagonist variants of the naturally-occurringhuman ABP and polypeptide fusions thereof. Fusions comprising additionalamino acids at the amino terminus, carboxyl terminus, or both, areencompassed by the term “antigen-binding polypeptide.” Exemplary fusionsinclude, but are not limited to, e.g., methionyl ABP in which amethionine is linked to the N-terminus of ABP resulting from therecombinant expression, fusions for the purpose of purification(including but not limited to, to poly-histidine or affinity epitopes),fusions for the purpose of linking ABP's to other biologically activemolecules, fusions with serum albumin binding peptides, and fusions withserum proteins such as serum albumin.

The term “antigen” or “binding partner” refers to a substance that isthe target for the binding activity exhibited by the ABP. Virtually anysubstance may be an antigen or binding partner for an ABP. Examples ofantigens or binding partners include, but are not limited to, Alpha-1antitrypsin, Angiostatin, Antihemolytic factor, antibodies,Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrialnatriuretic polypeptide, Atrial peptides, C—X—C chemokines (e.g.,T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1,PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractantprotein-1, Monocyte chemoattractant protein-2, Monocyte chemoattractantprotein-3, Monocyte inflammatory protein-1alpha, Monocyte inflammatoryprotein-1 beta, RANTES, I309, R83915, R91733, HCC1, T58847, D31065,T64262), CD40 ligand, C-kit Ligand, Collagen, Colony stimulating factor(CSF), Complement factor 5a, Complement inhibitor, Complement receptor1, cytokines, (e.g., epithelial Neutrophil Activating Peptide-78,GRO□/MGSA, GRQ, GRQ, MIP-1, MIP-1, MCP-1), Epidermal Growth Factor(EGF), Erythropoietin (“EPO”), Exfoliating toxins A and B, Factor IX,Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor (FGF),Fibrinogen, Fibronectin, G-CSF, GM-CSF, Glucocerebrosidase,Gonadotropin, growth factors, Hedgehog proteins (e.g., Sonic, Indian,Desert), Hemoglobin, Hepatocyte Growth Factor (HGF), Hirudin, Humanserum albumin, Insulin, Insulin-like Growth Factor (IGF), interferons(e.g., IFN-α, IFN-β, IFN-γ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, etc.), KeratinocyteGrowth Factor (KGF), Lactoferrin, leukemia inhibitory factor,Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), oncostatin M,Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones(e.g., Human Growth Hormone), Pleiotropin, Protein A, Protein G,Pyrogenic exotoxins A, B, and C, Relaxin, Renin, SCF, Soluble complementreceptor I, Soluble I-CAM 1, Soluble interleukin receptors (IL-1, 2, 3,4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15), Soluble TNF receptor,Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigens,i.e., Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED,SEE), Superoxide dismutase, Toxic shock syndrome toxin (TSST-1),Thymosin alpha 1, Tissue plasminogen activator, Tumor necrosis factorbeta (TNF beta), Tumor necrosis factor receptor (TNFR), Tumor necrosisfactor-alpha (TNF alpha), Vascular Endothelial Growth Factor (VEGEF),Urokinase and many others.

Many of these proteins are commercially available (See, e.g., the SigmaBioSciences 2002 catalogue and price list), and the correspondingprotein sequences and genes and, typically, many variants thereof, arewell-known (see, e.g., Genbank).

Additional antigens or binding partners include, but are not limited to,transcriptional and expression activators. Example transcriptional andexpression activators include genes and proteins that modulate cellgrowth, differentiation, regulation, or the like. Expression andtranscriptional activators are found in prokaryotes, viruses, andeukaryotes, including fungi, plants, and animals, including mammals,providing a wide range of therapeutic targets. It will be appreciatedthat expression and transcriptional activators regulate transcription bymany mechanisms, e.g., by binding to receptors, stimulating a signaltransduction cascade, regulating expression of transcription factors,binding to promoters and enhancers, binding to proteins that bind topromoters and enhancers, unwinding DNA, splicing pre-mRNA,polyadenylating RNA, and degrading RNA. Antigens or binding partnersinclude, but are not limited to, expression activators such ascytokines, inflammatory molecules, growth factors, their receptors, andoncogene products, e.g., interleukins (e.g., IL-1, IL-2, IL-8, etc.),interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF,SCF/c-Kit, CD40L/CD40, VLA-4VCAM-1, ICAM-1/LFA-1, and hyalurin/CD44;signal transduction molecules and corresponding oncogene products, e.g.,Mos, Ras, Raf, and Met; and transcriptional activators and suppressors,e.g., p53, Tat, Fos, Myc, Jun, Myb, Rel, and steroid hormone receptorssuch as those for estrogen, progesterone, testosterone, aldosterone, theLDL receptor ligand and corticosterone.

Vaccine proteins may be antigens or binding partners including, but notlimited to, proteins from infectious fungi, e.g., Aspergillus, Candidaspecies; bacteria, particularly E. coli, which serves a model forpathogenic bacteria, as well as medically important bacteria such asStaphylococci (e.g., aureus), or Streptococci (e.g., pneumoniae);protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba)and flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.);viruses such as (+) RNA viruses (examples include Poxviruses e.g.,vaccinia; Picornaviruses, e.g. polio; Togaviruses, e.g., rubella;Flaviviruses, e.g., HCV; and Coronaviruses), (−) RNA viruses (e.g.,Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses,e.g., influenza; Bunyaviruses; and Arenaviruses), dsDNA viruses(Reoviruses, for example), RNA to DNA viruses, i.e., Retroviruses, e.g.,HIV and HTLV, and certain DNA to RNA viruses such as Hepatitis B.

Antigens or binding partners may be enzymes including, but not limitedto, amidases, amino acid racemases, acylases, dehalogenases,dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases,esterases, isomerases, kinases, glucose isomerases, glycosidases,glycosyl transferases, haloperoxidases, monooxygenases (e.g., p450s),lipases, lignin peroxidases, nitrile hydratases, nitrilases, proteases,phosphatases, subtilisins, transaminase, and nucleases.

Agriculturally related proteins such as insect resistance proteins(e.g., the Cry proteins), starch and lipid production enzymes, plant andinsect toxins, toxin-resistance proteins, Mycotoxin detoxificationproteins, plant growth enzymes (e.g., Ribulose 1,5-BisphosphateCarboxylase/Oxygenase, “RUBISCO”), lipoxygenase (LOX), andPhosphoenolpyruvate (PEP) carboxylase may also be antigens or bindingpartners.

For example, the antigen or binding partner may be a disease-associatedmolecule, such as tumor surface antigen such as B-cell idiotypes, CD20on malignant B cells, CD33 on leukemic blasts, and HER2/neu on breastcancer. Alternatively, the antigen or binding partner may be a growthfactor receptor. Examples of the growth factors include, but are notlimited to, epidermal growth factors (EGFs), transferrin, insulin-likegrowth factor, transforming growth factors (TGFs), interleukin-1, andinterleukin-2. For example, a high expression of EGF receptors has beenfound in a wide variety of human epithelial primary tumors. TGF-α hasbeen found to mediate an autocrine stimulation pathway in cancer cells.Several murine monoclonal antibodies have been demonstrated to be ableto bind EGF receptors, block the binding of ligand to EGF receptors, andinhibit proliferation of a variety of human cancer cell lines in cultureand in xenograft medels. Mendelsohn and Baselga (1995) Antibodies togrowth factors and receptors, in Biologic Therapy of Cancer, 2nd Ed., JBLippincott, Philadelphia, pp 607-623. Thus, ABPs of the invention may beused to treat a variety of cancers.

The antigen or binding partner may also be cell surface protein orreceptor associated with coronary artery disease such as plateletglycoprotein Iib/IIIa receptor, autoimmune diseases such as CD4,CAMPATH-1 and lipid A region of the gram-negative bacteriallipopolysaccharide. Humanized antibodies against CD4 have been tested inclinical trials in the treatment of patients with mycosis fungoides,generalized postular psoriasis, severe psorisis, and rheumatoidarthritis. Antibodies against lipid A region of the gram-negativebacterial lipopolysaccharide have been tested clinically in thetreatment of septic shock. Antibodies against CAMPATH-1 have also beentested clinically in the treatment of against refractory rheumatoidarthritis. Thus, ABPs of the invention may be used to treat a variety ofautoimmune diseases. Vaswani et al. (1998) “Humanized antibodies aspotential therapeutic drugs” Annals of Allergy, Asthma and Immunology81:105-115.

The antigen or binding partner may also be proteins or peptidesassociated with human allergic diseases, such as inflammatory mediatorproteins, e.g. Interleukin-1 (IL-1), tumor necrosis factor (TNF),leukotriene receptor and 5-lipoxygenase, and adhesion molecules such asV-CAM/VLA-4. In addition, IgE may also serve as the antigen or bindingpartner because IgE plays pivotal role in type I immediatehypersensitive allergic reactions such as asthma. Studies have shownthat the level of total serum IgE tends to correlate with severity ofdiseases, especially in asthma. Burrows et al. (1989) “Association ofasthma with serum IgE levels and skin-test reactivity to allergens” NewEngl. L. Med. 320:271-277. Thus, ABPs selected against IgE may be usedto reduce the level of IgE or block the binding of IgE to mast cells andbasophils in the treatment of allergic diseases without havingsubstantial impact on normal immune functions.

The antigen or binding partner may also be a viral surface or coreprotein which may serve as an antigen to trigger immune response of thehost. Examples of these viral proteins include, but are not limited to,glycoproteins (or surface antigens, e.g., GP120 and GP41) and capsidproteins (or structural proteins, e.g., P24 protein); surface antigensor core proteins of hepatitis A, B, C, D or E virus (e.g. smallhepatitis B surface antigen (SHBsAg) of hepatitis B virus and the coreproteins of hepatitis C virus, NS3, NS4 and NS5 antigens); glycoprotein(G-protein) or the fusion protein (F-protein) of respiratory syncytialvirus (RSV); surface and core proteins of herpes simplex virus HSV-1 andHSV-2 (e.g., glycoprotein D from HSV-2).

The antigen or binding partner may also be a mutated tumor suppressorgene product that has lost its tumor-suppressing function and may renderthe cells more susceptible to cancer. Tumor suppressor genes are genesthat function to inhibit the cell growth and division cycles, thuspreventing the development of neoplasia. Mutations in tumor suppressorgenes cause the cell to ignore one or more of the components of thenetwork of inhibitory signals, overcoming the cell cycle check pointsand resulting in a higher rate of controlled cell growth—cancer.Examples of the tumor suppressor genes include, but are not limited to,DPC-4, NF-1, NF-2, RB, p53, WT1, BRCA1 and BRCA2.

DPC-4 is involved in pancreatic cancer and participates in a cytoplasmicpathway that inhibits cell division. NF-1 codes for a protein thatinhibits Ras, a cytoplasmic inhibitory protein. NF-1 is involved inneurofibroma and pheochromocytomas of the nervous system and myeloidleukemia. NF-2 encodes a nuclear protein that is involved in meningioma,schwanoma, and ependymoma of the nervous system. RB codes for the pRBprotein, a nuclear protein that is a major inhibitor of cell cycle. RBis involved in retinoblastoma as well as bone, bladder, small cell lungand breast cancer. p53 codes for p53 protein that regulates celldivision and can induce apoptosis. Mutation and/or inaction of p53 isfound in a wide ranges of cancers. WT1 is involved in Wilms tumor of thekidneys. BRCA1 is involved in breast and ovarian cancer, and BRCA2 isinvolved in breast cancer. Thus, ABPs may be used to block theinteractions of the gene product with other proteins or biochemicals inthe pathways of tumor onset and development.

The antigen or binding partner may be a CD molecule including but notlimited to, CD1a, CD1b, CD1c, CD1d, CD2, CD3γ, CD3δ, CD3ε, CD4, CD5,CD6, CD7, CD8α, CD8β, CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14,CD15, CD15s, CD16a, CD16b, CD18, CD19, CD20, CD21, CD22, CD23, CD24,CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36,CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44,CD45, CD45R, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f,CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61,CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d,CD66e, CD66f, CD67, CD68, CD69, CDw70, CD71, CD72, CD73, CD74, CDw75,CDw76, CD77, CD79α, CD79β, CD80, CD81, CD82, CD83, CD84, CD85, CD86,CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98,CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b,CDw108, CDw109, CD110-113, CD114, CD115, CD116, CD117, CD118, CD119,CD120a, CD120b, CD121a, CD121b, CD122, CD123, CDw124, CD125, CD126,CDw127, CDw128a, CDw128b, CD129, CDw130, CD131, CD132, CD133, CD134,CD135, CD136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143,CD144, CDw145, CD146, CD147, CD148, CDw149, CD150, CD151, CD152, CD153,CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164,CD165, CD166, and TCRζ. The antigen or binding partner may be VEGF, VEGFreceptor, EGFR, Her2, TNFa, TNFRI receptor, GPIIb/IIIa, IL-2R alphachain, IL-2R beta chain, RSV F protein, alpha4 integrin, IgE, IgEreceptor, digoxin, carpet viper venom, complement C5, OPGL, CA-125 tumorantigen, Staphylococci proteins, Staphylococcus epidermidis proteins,Staphylococcus aureus proteins, proteins involved Staphylococcalinfection (including but not limited to, Staphylococcus aureus andStaphylococcus epidermidis), IL-6 receptor, CTLA-4, RSV, Tac subunit ofIL-2 receptor, IL-5, and EpCam. The antigen or binding partner may be afragment of a molecule.

Examples of bispecific ABPs include, but are not limited to, those withone ABP directed against a tumor cell antigen and the other ABP directedagainst a cytotoxic trigger molecule such as anti-FcγRI/anti-CD 15,anti-p185^(HER2)/FcγRIII (CD16), anti-CD3/anti-malignant B-cell (1D10),anti-CD3/anti-p185^(HER2), anti-CD3/anti-p97, anti-CD3/anti-renal cellcarcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma),anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGFreceptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19,anti-CD3/MoV18, anti-neural cell adhesion molecule (NCAM)/anti-CD3,anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinomaassociated antigen (AMOC-31)/anti-CD3; bispecific ABPs with one ABPwhich binds specifically to a tumor antigen and another ABP which bindsto a toxin such as anti-saporin/anti-Id-1, anti-CD22/anti-saporin,anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin Achain, anti-interferon-α (IFN-α)/anti-hybridoma idiotype,anti-CEA/anti-vinca alkaloid; bispecific ABPs for converting enzymeactivated prodrugs such as anti-CD30/anti-alkaline phosphatase (whichcatalyzes conversion of mitomycin phosphate prodrug to mitomycinalcohol); bispecific ABPs which can be used as fibrinolytic agents suchas anti-fibrin/anti-tissue plasminogen activator (tPA),anti-fibrin/anti-urokinase-type plasminogen activator (uPA); bispecificABPs for targeting immune complexes to cell surface receptors such asanti-low density lipoprotein (LDL)/anti-Fc receptor (e.g. FcγRI, FcγRIIor FcγRIII); bispecific ABPs for use in therapy of infectious diseasessuch as anti-CD3/anti-herpes simplex virus (HSV), anti-T-cellreceptor:CD3 complex/anti-influenza, anti-FcγR/anti-HIV; bispecific ABPsfor tumor detection in vitro or in vivo such as anti-CEA/anti-EOTUBE,anti-CEA/anti-DPTA, anti-p185^(HER2)/anti-hapten; bispecific ABPs asvaccine adjuvants (see Fanger, M W et al., Crit. Rev Immunol. 1992;12(34):101-24, which is incorporated by reference herein); andbispecific ABPs as diagnostic tools such as anti-rabbitIgG/anti-ferritin, anti-horse radish peroxidase (HRP)/anti-hormone,anti-somatostatin/anti-substance P, anti-HRP/anti-FITC,anti-CEA/anti-β-galactosidase (see Nolan, O et R. O'Kennedy, BiochimBiophys Acta. 1990 Aug. 1; 1040(1):1-11, which is incorporated byreference herein). Examples of trispecific ABPs includeanti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 andanti-CD3/anti-CD8/anti-CD37.

Various references disclose modification of polypeptides by polymerconjugation or glycosylation. The term “ABP” or “antigen-bindingpolypeptide” includes, but is not limited to, polypeptides conjugated toa polymer such as PEG and may be comprised of one or more additionalderivitizations of cysteine, lysine, N or C-terminal amino acids, orother residues. In addition, the ABP may comprise a linker, polymer orbiologically active molecule, wherein the amino acid to which thelinker, polymer, or biologically active molecule is conjugated may be anon-natural amino acid according to the present invention, or may beconjugated to a naturally encoded amino acid utilizing techniques knownin the art such as coupling to lysine or cysteine. U.S. Pat. No.4,904,584 discloses PEGylated lysine depleted polypeptides, wherein atleast one lysine residue has been deleted or replaced with any otheramino acid residue. WO 99/67291 discloses a process for conjugating aprotein with PEG, wherein at least one amino acid residue on the proteinis deleted and the protein is contacted with PEG under conditionssufficient to achieve conjugation to the protein. WO 99/03887 disclosesPEGylated variants of polypeptides belonging to the growth hormonesuperfamily, wherein a cysteine residue has been substituted with anon-essential amino acid residue located in a specified region of thepolypeptide. WO 00/26354 discloses a method of producing a glycosylatedpolypeptide variant with reduced allergenicity, which as compared to acorresponding parent polypeptide comprises at least one additionalglycosylation site.

The term “antigen-binding polypeptide” also includes glycosylated ABP's,such as but not limited to, polypeptides glycosylated at any amino acidposition, N-linked or O-linked glycosylated forms of the polypeptide.Variants containing single nucleotide changes are also considered asbiologically active variants of ABP. In addition, splice variants arealso included. The term “antigen-binding polypeptide” also includes ABPheterodimers, homodimers, heteromultimers, or homomultimers of any oneor more ABP or any other polypeptide, protein, carbohydrate, polymer,small molecule, linker, ligand, or other biologically active molecule ofany type, linked by chemical means or expressed as a fusion protein, aswell as polypeptide analogues containing, for example, specificdeletions or other modifications yet maintain biological activity.

In some embodiments, the antigen-binding polypeptides further comprisean addition, substitution or deletion that modulates biological activityof the ABP. For example, the additions, substitutions or deletions maymodulate one or more properties or activities of the ABP, including butnot limited to, modulating affinity for the antigen, modulate (includingbut not limited to, increases or decreases) antigen conformational orother secondary, tertiary or quaternary structural changes, stabilizeantigen conformational or other secondary, tertiary or quaternarystructural changes, induce or cause antigen conformational or othersecondary, tertiary or quaternary structural changes, modulatecirculating half-life, modulate therapeutic half-life, modulatestability of the polypeptide, modulate dose, modulate release orbio-availability, facilitate purification, or improve or alter aparticular route of administration. Similarly, antigen-bindingpolypeptides may comprise protease cleavage sequences, reactive groups,antibody-binding domains (including but not limited to, FLAG orpoly-His) or other affinity based sequences (including but not limitedto, FLAG, poly-His, GST, etc.) or linked molecules (including but notlimited to, biotin) that improve detection (including but not limitedto, GFP), purification or other traits of the polypeptide.

The term “antigen-binding polypeptide” also encompasses ABP homodimers,heterodimers, homomultimers, and heteromultimers that are linked,including but not limited to those linked directly via non-naturallyencoded amino acid side chains, either to the same or differentnon-naturally encoded amino acid side chains, to naturally-encoded aminoacid side chains, as fusions, or indirectly via a linker. Exemplarylinkers include but are not limited to, small organic compounds, watersoluble polymers of a variety of lengths such as poly(ethylene glycol)or polydextran, or polypeptides of various lengths.

Those of skill in the art will appreciate that amino acid positionscorresponding to positions in a particular antigen-binding polypeptidesequence can be readily identified in a fragment of the antigen-bindingpolypeptide or related antigen-binding polypeptide, etc. For example,sequence alignment programs such as BLAST can be used to align andidentify a particular position in a protein that corresponds with aposition in a related sequence.

The term “antigen-binding polypeptide” encompasses antigen-bindingpolypeptides comprising one or more amino acid substitutions, additionsor deletions. Antigen-binding polypeptides of the present invention maybe comprised of modifications with one or more natural amino acids inconjunction with one or more non-natural amino acid modification.Exemplary substitutions in a wide variety of amino acid positions innaturally-occurring ABP polypeptides have been described, including butnot limited to substitutions that modulate one or more of the biologicalactivities of the antigen-binding polypeptide, such as but not limitedto, increase agonist activity, increase solubility of the polypeptide,convert the polypeptide into an antagonist, etc. and are encompassed bythe term “ABP.”

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the 20 common amino acids or pyrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrolysine and selenocysteine) but are not themselves naturallyincorporated into a growing polypeptide chain by the translationcomplex. Examples of such non-naturally-occurring amino acids include,but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include, but are not limited to, various water soluble polymers,peptides or proteins such as serum albumin, or other moieties thatincrease serum half-life of peptides.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages mean thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meanthat the linkage can be degraded by one or more enzymes. As understoodin the art, PEG and related polymers may include degradable linkages inthe polymer backbone or in the linker group between the polymer backboneand one or more of the terminal functional groups of the polymermolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide. Branched linkersmay be used in antigen-binding polypeptides of the invention.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include, butare not limited to, any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs, harddrugs, soft drugs, dyes, lipids, nucleosides, oligonucleotides, toxins,cells, viruses, liposomes, microparticles and micelles. Classes ofbiologically active agents that are suitable for use with the inventioninclude, but are not limited to, drugs, prodrugs, radionuclides, imagingagents, polymers, antibiotics, fungicides, anti-viral agents,anti-inflammatory agents, anti-tumor agents, cardiovascular agents,anti-anxiety agents, hormones, growth factors, steroidal agents,microbially derived toxins, and the like.

In certain embodiments, the ABP molecules of this invention can be usedto direct biologically active molecules or detectable labels to a tumorsite. This can facilitate tumor killing, detection and/or localizationor other effect. Diagnostic probes or imaging probes may also be linkedto ABP molecules of the invention. In certain particularly preferredembodiments, the biologically active molecule component of the ABP is a“radiopaque” label, e.g. a label that can be easily visualized using forexample x-rays. Radiopaque materials are well known to those of skill inthe art. The most common radiopaque materials include iodide, bromide orbarium salts. Other radiopaque materials are also known and include, butare not limited to organic bismuth derivatives (see, e.g., U.S. Pat. No.5,939,045), radiopaque multiurethanes (see U.S. Pat. No. 5,346,981),organobismuth composites (see, e.g., U.S. Pat. No. 5,256,334),radiopaque barium multimer complexes (see, e.g., U.S. Pat. No.4,866,132), and the like.

The ABP's of this invention can be coupled directly to the radiopaquemoiety or they can be attached to a “package” (e.g. a chelate, aliposome, a multimer microbead, etc.) carrying or containing theradiopaque material.

In addition to radioopaque labels, other labels are also suitable foruse in this invention. Detectable labels suitable for use as thebiologically active molecule component of the ABP's of this inventioninclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include magnetic beads (e.g.Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, texasred, rhodamine, green fluorescent protein, and the like), radiolabels(e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radishperoxidase, alkaline phosphatase and others commonly used in an ELISA),and colorimetric labels such as colloidal gold or colored glass orplastic (e.g. multistyrene, multipropylene, latex, etc.) beads.

Various preferred radiolabels include, but are not limited to ⁹⁹Tc,²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ¹¹³mIn, ⁹⁷Ru, ⁶²Cu, ⁶⁴¹Cu, ⁵²Fe, ⁵²mMn,⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr,¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd,¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, and ¹¹¹Ag.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm, scintillation detectors, and the like. Fluorescent markers may bedetected using a photodetector to detect emitted illumination. Enzymaticlabels are typically detected by providing the enzyme with a substrateand detecting the reaction product produced by the action of the enzymeon the substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

In certain specific embodiments, this invention contemplates the use ofimmunoconjugates (chimeric moieties) for the detection of tumors and/orother cancer cells. Thus, for example, the bispecific antibodies of thisinvention can be conjugated to gamma-emitting radioisotopes (e.g.,Na-22, Cr-51, Co-60, Tc-99, I-125, I-131, Cs-137, Ga-67, Mo-99) fordetection with a gamma camera, to positron emitting isotopes (e.g. C-11,N-13, O-15, F-18, and the like) for detection on a Positron EmissionTomography (PET) instrument, and to metal contrast agents (e.g., Gdcontaining reagents, Eu containing reagents, and the like) for magneticresonance imaging (MRI), In addition, the bispecific antibodies of thisinvention can be used in traditional immunohistochemistry (e.g.fluorescent labels, nanocrystal labels, enzymatic and colormetric labelsetc.).

In another embodiment, the biologically active molecule can be aradiosensitizer that enhances the cytotoxic effect of ionizing radiation(e.g., such as might be produced by ⁶⁰Co or an x-ray source) on a cell.Numerous radiosensitizing agents are known and include, but are notlimited to benzoporphyrin derivative compounds (see, e.g., U.S. Pat. No.5,945,439), 1,2,4-benzotriazine oxides (see, e.g., U.S. Pat. No.5,849,738), compounds containing certain diamines (see, e.g., U.S. Pat.No. 5,700,825), BCNT (see, e.g., U.S. Pat. No. 5,872,107),radiosensitizing nitrobenzoic acid amide derivatives (see, e.g., U.S.Pat. No. 4,474,814), various heterocyclic derivatives (see, e.g., U.S.Pat. No. 5,064,849), platinum complexes (see, e.g., U.S. Pat. No.4,921,963), and the like.

The biologically active molecule may also be a ligand, an epitope tag, apeptide, a protein, or another ABP. Ligand and antibodies may be thosethat bind to surface markers on immune cells. Chimeric moleculesutilizing such antibodies as biologically active molecules act asbifunctional linkers establishing an association between the immunecells bearing binding partner for the ligand or ABP and the tumor cellsexpressing the EGFR family member(s).

Many of the pharmaceuticals and/or radiolabels described herein may beprovided as a chelate, particularly where a pre-targeting strategy isutilized. The chelating molecule is typically coupled to a molecule(e.g. biotin, avidin, streptavidin, etc.) that specifically binds anepitope tag attached to the bispecific and/or multispecific ABP.

Chelating groups are well known to those of skill in the art. In certainembodiments, chelating groups are derived from ethylene diaminetetra-acetic acid (EDTA), diethylene triamine penta-acetic acid (DTPA),cyclohexyl 1,2-diamine tetra-acetic acid (CDTA),ethyleneglycol-O,O′-bis(−2-aminoethyl)-N,N,N′,N′-tetra-acetic acid(EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid(HBED), triethylene tetramine hexa-acetic acid (TTHA),1,4,7,10-tetraazacyclododecane-N,N′-,N″,N′″-tetra-acetic acid (DOTA),hydroxyethyldiamine triacetic acid (HEDTA),1,4,8,11-tetra-azacyclotetradecane-N,N′,N″,N′″-tetra-acetic acid (TETA),substituted DTPA, substituted EDTA, and the like.

Examples of certain preferred chelators include unsubstituted or,substituted 2-iminothiolanes and 2-iminothiacyclohexanes, in particular2-imino-4-mercaptomethylthiolane, and SAPS(N-(4-[211At]astatophenethyl)succinimate).

One chelating agent, 1,4,7,10-tetraazacyclododecane-N,N,N″,N′″-tetraacetic acid (DOTA), is of particular interest because of itsability to chelate a number of diagnostically and therapeuticallyimportant metals, such as radionuclides and radiolabels.

Conjugates of DOTA and proteins such as antibodies have been described.For example, U.S. Pat. No. 5,428,156 teaches a method for conjugatingDOTA to antibodies and ABP fragments. To make these conjugates, onecarboxylic acid group of DOTA is converted to an active ester which canreact with an amine or sulfhydryl group on the ABP or ABP fragment.Lewis et al. (1994) Bioconjugate Chem. 5: 565-576, describes a similarmethod wherein one carboxyl group of DOTA is converted to an activeester, and the activated DOTA is mixed with an ABP, linking the ABP toDOTA via the epsilon-amino group of a lysine residue of the ABP, therebyconverting one carboxyl group of DOTA to an amide moiety.

Alternatively the chelating agent can be coupled, directly or through alinker, to an epitope tag or to a moiety that binds an epitope tag.Conjugates of DOTA and biotin have been described (see, e.g., Su (1995)J. Nucl. Med., 36 (5 Suppl): 154P, which discloses the linkage of DOTAto biotin via available amino side chain biotin derivatives such asDOTA-LC-biotin or DOTA-benzyl-4-(6-amino-caproamide)-biotin). Yau etal., WO 95/15335, disclose a method of producing nitro-benzyl-DOTAcompounds that can be conjugated to biotin. The method comprises acyclization reaction via transient projection of a hydroxy group;tosylation of an amine; deprotection of the transiently protectedhydroxy group; tosylation of the deprotected hydroxy group; andintramolecular tosylate cyclization. Wu et al. (1992) Nucl. Med. Biol.,19(2): 239-244 discloses a synthesis of macrocylic chelating agents forradiolabeling proteins with ¹¹¹IN and ⁹⁰Y. Wu et al. makes a labeledDOTA-biotin conjugate to study the stability and biodistribution ofconjugates with avidin, a model protein for studies. This conjugate wasmade using a biotin hydrazide which contained a free amino group toreact with an in situ generated activated DOTA derivative.

ABP's of this invention may be fused to other biologically activemolecules, including, but are not limited to, cytotoxic drugs, toxins,peptides, proteins, enzymes and viruses (Chester, (2000) Dis. Markers16:53-62; Rippmann et al. Biochem J. (2000) Biochem J. 349 (Pt.3):805-812, Kreitman, R. J. (2001) Curr. Pharm. Biotechnol. 2:313-325;Rybak, S. M. (2001) Expert Opin. Biol. Ther. 1:995-1003; van Beusechem,V. W. et al. J. Virol. (2002) 76:2753-2762).

A potent cytotoxic agent, or payload, may be bound to ABP's that targetand bind to antigens that are found predominantly on target cells(including but not limited to, cancer cells). The payload agent islinked to the ABP via a link that is stable in the bloodstream, or maybe susceptible to cleavage under conditions present at, for example, thetumor site. Payload agents such as toxins are delivered to target cellsand thus cell killing can be initiated via a mechanism dependent on thetoxin.

Examples of such toxins include, but are not limited to, small moleculessuch as fungal derived calicheamicins (Hinman et al. (1993) Cancer Res.53: 3336-3342) and maytansinoids (Liu et al. (1996) PNAS USA93:8618-8623, Smith, S. (2001) Curr. Opin. Mol. Ther. 3(2):198-203),trichothene, and CC 1065, or proteins, e.g. ricin A chain (Messman, etal. (2000) Clin. Cancer Res. 6(4):1302-1313), Pseudomonas exotoxin (Turet al. (2001) Intl. J. Mol. Med. 8(5):579-584), diphtheria toxin(LeMaistre et al. (1998) Blood 91(2):399-405), and ribosome-inactivatingproteins (Tazzari, et al. (2001), J. Immunol. 167:4222-4229). In aspecific embodiment, one or more calicheamicin molecules may be used.Members of the calicheamicin family of antibiotics are capable ofproducing double-stranded DNA breaks at sub-picomolar concentrations.Structured analogues of calicheamicin are also known. See Hinman et al.,Cancer Research 53: 3336-42 (1993); Lode et al. (1998) Cancer Research58:2925-28. An example of an immunotoxin that has gained FDA approval isMylotarg® (Wyeth Ayerst), a calichaemicin-conjugated anti-CD33 for acutemyelogenous leukemia (Sievers et al. (1999) Blood 93(11):3678-3684;Bernstein (2000) Leukemia 14:474-475). In a similar fashion, ABP's ofthis invention may be fused to toxins. Alternatively, ABP's of theinvention may be fused with botulinum A neurotoxin, a protein complexproduced by the bacterium Clostridium botulinum.

In yet another embodiment, the ABP's of the invention may comprise oneor more enzymatically active toxins and/or fragments thereof. Examplesof such toxins include non-binding active fragments of diphtheria toxin,diphtheria A chain, exotoxin A chain (from Pseudomonas aeruginosa),ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, dianthinproteins, Phytolaca americana proteins (PAPI, PAPAII, and PAP-S),momordica charantia inhibitor, curcin, crotin sapaonaria, officinalisinhibitor, gelonin, mitogellin, restrictoein, phenomycin, enomycin, andthe tricothecenes. See e.g., WO 93/21232. Particularly preferredcytotoxins include Pseudomonas exotoxins (PE), Diphtheria toxins, ricin,and abrin. Pseudomonas exotoxin and Dipthteria toxin are well known.Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylatingelongation factor 2 thereby inhibiting protein synthesis. Additionalcitations regarding immunotoxins include Brinkmann, U. (2000) In Vivo14:21-28, Niv et al. (2001) Curr. Pharm. Biotechnol. 2:19-46, Reiter etal. (2001) Adv. Cancer Res. 81:93-124, Kreitman, R. J. (1999) Curr.Opin. Immunol., 11:570-578; Hall (2001) Meth. Mol. Biol. 166:139-154;Kreitman (2001) Curr. Opin. Investig. Drugs 2(9):1282-1293. Methods ofcloning genes encoding PE or DT fused to various ligands are well knownto those of skill in the art (see, e.g., Siegall et al. (1989) FASEB J.,3: 2647-2652; and Chaudhary et al. (1987) Proc. Natl. Acad. Sci. USA,84: 4538-4542). All citations are incorporated by reference herein.

Other suitable biologically active molecules include pharmacologicalagents or encapsulation systems containing various pharmacologicalagents. Thus, the targeting molecule of the chimeric molecule may beattached directly to a drug that is to be delivered directly to thetumor. Such drugs are well known to those of skill in the art andinclude, but are not limited to, doxorubicin, vinblastine, genistein, anantisense molecule, and the like.

Alternatively, the biologically active molecule may be an encapsulationsystem, such as a viral capsid, a liposome, or micelle that contains atherapeutic composition such as a drug, a nucleic acid (e.g. anantisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art. See, for example, U.S. Pat. No. 4,957,735,Connor et al. (1985) Pharm. Ther., 28: 341-365. Due to their antigenspecificity, ABP's of the invention may be used to direct drug-loadedliposomes to their target. See Park, J. W. et al. (2002) Clin. CancerRes. 8, 1172-1181 and Shi, N. et al (2001) Pharm. Res. 18, 1091-1095.

ABP's of the invention may be conjugated to molecules such as PEG toimprove in vivo delivery and pharmacokinetic profiles. Leong et al.describe site-specific PEGylation of a Fab′ fragment of an anti-IL-8antibody with a decreased clearance rate over the non-PEGylated form andlittle or no loss of antigen binding activity (Leong, S. R. et al.(2001) Cytokine 16:106-119).

The ABP's of the present invention may be linked to a prodrug. The term“prodrug” as used herein means a pharmacologically inactive, or reducedactivity, derivative of an active drug. Prodrugs may be designed tomodulate the amount of a drug or biologically active molecule thatreaches a desired site of action through the manipulation of theproperties of a drug, such as physicochemical, biopharmaceutical, orpharmacokinetic properties. Prodrugs are converted into active drugwithin the body through enzymatic or non-enzymatic reactions. Prodrugsmay provide improved physicochemical properties such as bettersolubility, enhanced delivery characteristics, such as specificallytargeting a particular cell, tissue, organ or ligand, and improvedtherapeutic value of the drug. ABP's of the invention may be fused toenzymes for prodrug activation (Kousparou, C. A., et al. (2002) Int. J.Cancer 99, 138-148). (2002) Recombinant molecules may comprise an ABPand an enzyme that acts upon a prodrug to release a cytotoxin such ascyanide.

The therapeutic agents may be administered as a prodrug and subsequentlyactivated by a prodrug-activating enzyme that converts a prodrug likepeptidyl chemotherapeutic agent to an active anti-cancer drug. See,e.g., WO 88/07378; WO 81/01145; U.S. Pat. No. 4,975,278. In general, theenzyme component includes any enzyme capable of acting on a prodrug insuch a way as to convert it into its more active, cytotoxic form.

Enzymes that may be useful include, but are not limited to, alkalinephosphatase useful for converting phosphate-containing prodrugs intofree drugs, arylsulfatase useful for converting sulfate containingprodrugs into free drugs; cytosine deaminase useful for convertingnon-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidases and cathepsins (such as cathepsins B and L), that areuseful for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, useful for converting prodrugs that containD-amino acid substituents; carbohydrate cleaving enzymes such asβ-galactosidase and neuraminidase useful for converting glycosylatedprodrugs into free drugs; β-lactamase useful for converting drugsderivatized with β-lactams into free drugs; and penicillin amidases,such as penicillin V amidase or penicillin G amidase, useful forconverting drugs derivatized at their amino nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs.

Alternatively, antibodies with enzymatic activity, also known in the artas “abzymes,” may be used to convert the prodrugs of the invention intofree active drugs. See e.g., Massey, (1987) 328:457-48.

One of skill will appreciate that the bispecific and/or multispecificABP of this invention and the biologically active molecule moieties cantypically be joined together in any order. Thus, for example, where thetargeting molecule is a single chain protein the biologically activemolecule may be joined to either the amino or carboxy termini of thetargeting molecule. The biologically active molecule can also be joinedto an internal region of the bispecific and/or multispecific ABP, orconversely. Similarly, the bispecific and/or multispecific ABP can bejoined to an internal location or a terminus of the biologically activemolecule. In any case, attachment points are selected that do notinterfere with the respective activities of the bispecific and/ormultispecific ABP or the biologically active molecule.

The bispecific and/or multispecific ABP and the biologically activemolecule can be attached by any of a number of means well known to thoseof skill in the art. Typically the biologically active molecule isconjugated, either directly or through a linker (spacer), to thebispecific ABP. However, where both the biologically active molecule andthe bispecific ABP are both polypeptides it may be desired torecombinantly express the chimeric molecule as a single-chain fusionprotein.

In one embodiment, the bispecific and/or multispecific ABP is chemicallyconjugated to the biologically active molecule (e.g., a cytotoxin, alabel, a ligand, a drug, an ABP, a liposome, etc.). Means of chemicallyconjugating molecules are well known to those of skill in the art.

The procedure for attaching an agent to an ABP or other polypeptidetargeting molecule will vary according to the chemical structure of theagent. Polypeptides typically contain variety of functional groups;e.g., carboxylic acid (COOH) or free amine (—NH₂) groups, which areavailable for reaction with a suitable functional group on abiologically active molecule to bind the biologically active moleculethereto.

Alternatively, the bispecific ABP and/or biologically active moleculecan be derivatized to expose or attach additional reactive functionalgroups. The derivatization can involve attachment of any of a number oflinker molecules such as those available from Pierce Chemical Company,Rockford, Ill.

In some circumstances, it may be desirable to free the biologicallyactive molecule from the bispecific and/or multispecific ABP, oractivate a prodrug, when the chimeric moiety has reached its targetsite. Therefore, chimeric conjugates comprising linkages that arecleavable in the vicinity of the target site can be used when thebiologically active molecule is to be released at the target site.Cleaving of the linkage to release the agent from the ABP may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (e.g. whenexposed to tumor-associated enzymes or acidic pH) may be used.

A number of different cleavable linkers are known to those of skill inthe art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. Themechanisms for release of an agent from these linker groups include, forexample, irradiation of a photolabile bond and acid-catalyzedhydrolysis. U.S. Pat. No. 4,671,958, for example, includes a descriptionof immunoconjugates comprising linkers which are cleaved at the targetsite in vivo by the proteolytic enzymes of the patient's complementsystem. The length of the linker may be predetermined or selecteddepending upon a desired spatial relationship between the ABP and themolecule linked to it. In view of the large number of methods that havebeen reported for attaching a variety of radiodiagnostic compounds,radiotherapeutic compounds, drugs, toxins, and other agents toantibodies one skilled in the art will be able to determine a suitablemethod for attaching a given agent to an ABP or other polypeptide.

In certain embodiments, the biologically active molecule comprises achelate that is attached to an ABP or to an epitope tag. The bispecificand/or multispecific ABP bears a corresponding epitope tag or ABP sothat simple contacting of the bispecific and/or multispecific ABP to thechelate results in attachment of the ABP to the biologically activemolecule. The combining step can be performed after the moiety is used(pretargeting strategy) or the target tissue can be bound to thebispecific and/or multispecific ABP before the chelate is delivered.Methods of producing chelates suitable for coupling to various targetingmoieties are well known to those of skill in the art (see, e.g., U.S.Pat. Nos. 6,190,923, 6,187,285, 6,183,721, 6,177,562, 6,159,445,6,153,775, 6,149,890, 6,143,276, 6,143,274, 6,139,819, 6,132,764,6,123,923, 6,123,921, 6,120,768, 6,120,751, 6,117,412, 6,106,866,6,096,290, 6,093,382, 6,090,800, 6,090,408, 6,088,613, 6,077,499,6,075,010, 6,071,494, 6,071,490, 6,060,040, 6,056,939, 6,051,207,6,048,979, 6,045,821, 6,045,775, 6,030,840, 6,028,066, 6,022,966,6,022,523, 6,022,522, 6,017,522, 6,015,897, 6,010,682, 6,010,681,6,004,533, and 6,001,329).

Where the bispecific and/or multispecific ABP and/or the biologicallyactive molecule are both single chain proteins and relatively short(i.e., less than about 50 amino acids) they can be synthesized usingstandard chemical peptide synthesis techniques. Where both componentsare relatively short, the chimeric moiety can be synthesized as a singlecontiguous polypeptide. Alternatively, a bispecific and/or multispecificABP and the biologically active molecule may be synthesized separatelyand then fused by condensation of the amino terminus of one moleculewith the carboxyl terminus of the other molecule thereby forming apeptide bond. Alternatively, the bispecific and/or multispecific ABP andbiologically active molecules may each be condensed with one end of apeptide spacer molecule thereby forming a contiguous fusion protein.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), andStewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill. (1984).

A “bifunctional polymer” refers to a polymer comprising two discretefunctional groups that are capable of reacting specifically with othermoieties (including but not limited to, amino acid side groups) to formcovalent or non-covalent linkages. A bifunctional linker having onefunctional group reactive with a group on a particular biologicallyactive component, and another group reactive with a group on a secondbiological component, may be used to form a conjugate that includes thefirst biologically active component, the bifunctional linker and thesecond biologically active component. Many procedures and linkermolecules for attachment of various compounds to peptides are known.See, e.g., European Patent Application No. 188,256; U.S. Pat. Nos.4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and4,589,071 which are incorporated by reference herein. A“multi-functional polymer” refers to a polymer comprising two or morediscrete functional groups that are capable of reacting specificallywith other moieties (including but not limited to, amino acid sidegroups) to form covalent or non-covalent linkages. A bi-functionalpolymer or multi-functional polymer may be any desired molecular lengthor molecular weight, and may be selected to provide a particular desiredspacing or conformation between one of molecules linked to the ABP.

Where substituent groups are specified by their conventional chemicalformulas, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, for example, the structure —CH₂O— isequivalent to the structure —OCH₂—.

The term “substituents” includes but is not limited to “non-interferingsubstituents”. “Non-interfering substituents” are those groups thatyield stable compounds. Suitable non-interfering substituents orradicals include, but are not limited to, halo, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₁-C₁₂ aralkyl, C₁-C₁₂ alkaryl,C₃-C₁₂ cycloalkyl, C₃-C₁₂ cycloalkenyl, phenyl, substituted phenyl,toluoyl, xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₂-C₁₂ alkoxyaryl,C₇-C₁₂ aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀alkylsulfonyl, —(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8,aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclicradical, substituted heterocyclic radical, nitroalkyl, —NO₂, —CN,—NRC(O)—(C₁-C₁₀ alkyl), —C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkyl thioalkyl,—C(O)O—(C₁-C₁₀ alkyl), —OH, —SO₂, ═S, —COOH, —NR₂, carbonyl,—C(O)—(C₁-C₁₀ alkyl)-CF3, —C(O)—CF3, —C(O)NR2, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀aryl), —C(O)—(C₁-C₁₀ aryl), —(CH₂)_(m)—O—(—(CH₂)_(m)—O—(C₁-C₁₀ alkyl)wherein each m is from 1 to 8, —C(O)NR₂, —C(S)NR₂, —SO₂NR₂, —NRC(O)NR₂,—NRC(S)NR₂, salts thereof, and the like. Each R as used herein is H,alkyl or substituted alkyl, aryl or substituted aryl, aralkyl, oralkaryl.

The term “halogen” includes fluorine, chlorine, iodine, and bromine.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by the structures —CH₂CH₂— and —CH₂CH₂CH₂CH₂—, and furtherincludes those groups described below as “heteroalkylene.” Typically, analkyl (or alkylene) group will have from 1 to 24 carbon atoms, withthose groups having 10 or fewer carbon atoms being preferred in thepresent invention. A “lower alkyl” or “lower alkylene” is a shorterchain alkyl or alkylene group, generally having eight or fewer carbonatoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, the same or different heteroatoms can also occupyeither or both of the chain termini (including but not limited to,alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino,aminooxyalkylene, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkylor heterocycloalkyl include saturated and unsaturated ring linkages.Additionally, for heterocycloalkyl, a heteroatom can occupy the positionat which the heterocycle is attached to the remainder of the molecule.Examples of cycloalkyl include, but are not limited to, cyclopentyl,cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.Examples of heterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. Additionally, the termencompasses bicyclic and tricyclic ring structures. Similarly, the term“heterocycloalkylene” by itself or as part of another substituent meansa divalent radical derived from heterocycloalkyl, and the term“cycloalkylene” by itself or as part of another substituent means adivalent radical derived from cycloalkyl.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto ABP can result in changes including, but not limited to, increased ormodulated serum half-life, or increased or modulated therapeutichalf-life relative to the unmodified form, modulated immunogenicity,modulated physical association characteristics such as aggregation andmultimer formation, altered receptor binding and altered receptordimerization or multimerization. The water soluble polymer may or maynot have its own biological activity, and may be utilized as a linkerfor attaching an ABP to other substances, including but not limited toone or more ABP's, or one or more biologically active molecules.Suitable polymers include, but are not limited to, polyethylene glycol,polyethylene glycol propionaldehyde, mono C1-C10 alkoxy or aryloxyderivatives thereof (described in U.S. Pat. No. 5,252,714 which isincorporated by reference herein), monomethoxy-polyethylene glycol,polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylethermaleic anhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextranderivatives including dextran sulfate, polypropylene glycol,polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol,heparin, heparin fragments, polysaccharides, oligosaccharides, glycans,cellulose and cellulose derivatives, including but not limited tomethylcellulose and carboxymethyl cellulose, starch and starchderivatives, polypeptides, polyalkylene glycol and derivatives thereof,copolymers of polyalkylene glycols and derivatives thereof, polyvinylethyl ethers, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, andthe like, or mixtures thereof. Examples of such water soluble polymersinclude, but are not limited to, polyethylene glycol and serum albumin.

As used herein, the term “polyalkylene glycol” or “poly(alkene glycol)”refers to polyethylene glycol (poly(ethylene glycol)), polypropyleneglycol, polybutylene glycol, and derivatives thereof. The term“polyalkylene glycol” encompasses both linear and branched polymers andaverage molecular weights of between 0.1 kDa and 100 kDa. Otherexemplary embodiments are listed, for example, in commercial suppliercatalogs, such as Shearwater Corporation's catalog “Polyethylene Glycoland Derivatives for Biomedical Applications” (2001).

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (preferably from 1 to 3 rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(including but not limited to, aryloxy, arylthioxy, arylalkyl) includesboth aryl and heteroaryl rings as defined above. Thus, the term“arylalkyl” is meant to include those radicals in which an aryl group isattached to an alkyl group (including but not limited to, benzyl,phenethyl, pyridylmethyl and the like) including those alkyl groups inwhich a carbon atom (including but not limited to, a methylene group)has been replaced by, for example, an oxygen atom (including but notlimited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,and the like).

Each of the above terms (including but not limited to, “alkyl,”“heteroalkyl,” “aryl” and “heteroaryl”) are meant to include bothsubstituted and unsubstituted forms of the indicated radical. Exemplarysubstituents for each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such a radical. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, but are not limited to: halogen, —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″ and R″″are independently selected from hydrogen, alkyl, heteroalkyl, aryl andheteroaryl. When a compound of the invention includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

As used herein, the term “modulated serum half-life” means the positiveor negative change in circulating half-life of a modified ABP relativeto its non-modified form. Serum half-life is measured by taking bloodsamples at various time points after administration of ABP, anddetermining the concentration of that molecule in each sample.Correlation of the serum concentration with time allows calculation ofthe serum half-life. Increased serum half-life desirably has at leastabout two-fold, but a smaller increase may be useful, for example whereit enables a satisfactory dosing regimen or avoids a toxic effect. Insome embodiments, the increase is at least about three-fold, at leastabout five-fold, or at least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the half-life of the therapeuticallyeffective amount of an ABP or ABP comprising a modified biologicallyactive molecule, relative to its non-modified form. Therapeutichalf-life is measured by measuring pharmacokinetic and/orpharmacodynamic properties of the molecule at various time points afteradministration. Increased therapeutic half-life desirably enables aparticular beneficial dosing regimen, a particular beneficial totaldose, or avoids an undesired effect. In some embodiments, the increasedtherapeutic half-life results from increased potency, increased ordecreased binding of the modified molecule to its target, or an increaseor decrease in another parameter or mechanism of action of thenon-modified molecule.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is substantially free of other cellularcomponents with which it is associated in the natural state. It can bein a homogeneous state. Isolated substances can be in either a dry orsemi-dry state, or in solution, including but not limited to, an aqueoussolution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it means that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes8:91-98 (1994)).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally encoded amino acid. As used herein, the terms encompassamino acid chains of any length, including full length proteins (i.e.,antigens), wherein the amino acid residues are linked by covalentpeptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 60% identity,optionally about 65%, about 70%, about 75%, about 80%, about 85%, about90%, or about 95% identity over a specified region), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Thisdefinition also refers to the complement of a test sequence. Theidentity can exist over a region that is at least about 50 amino acidsor nucleotides in length, or over a region that is 75-100 amino acids ornucleotides in length, or, where not specified, across the entiresequence or a polynucleotide or polypeptide.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, including but not limited to, by thelocal homology algorithm of Smith and Waterman (1970) Adv. Appl. Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.); or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information. TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989)Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTalgorithm is typically performed with the “low complexity” filter turnedoff.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditions oflow ionic strength and high temperature as is known in the art.Typically, under stringent conditions a probe will hybridize to itstarget subsequence in a complex mixture of nucleic acid (including butnot limited to, total cellular or library DNA or RNA) but does nothybridize to other sequences in the complex mixture. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions maybe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (including but not limited to, 10 to 50 nucleotides) and atleast about 60° C. for long probes (including but not limited to,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide. Forselective or specific hybridization, a positive signal may be at leasttwo times background, optionally 10 times background hybridization.Exemplary stringent hybridization conditions can be as following: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. Suchwashes can be performed for 5, 15, 30, 60, 120, or more minutes.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya such as animals (including but notlimited to, mammals, insects, reptiles, birds, etc.), ciliates, plants(including but not limited to, monocots, dicots, algae, etc.), fungi,yeasts, flagellates, microsporidia, protists, etc.

As used herein, the term “non-eukaryote” refers to non-eukaryoticorganisms. For example, a non-eukaryotic organism can belong to theEubacteria (including but not limited to, Escherichia coli, Thermusthermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens,Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain,or the Archaea (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.)phylogenetic domain.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human, who is the object of treatment,observation or experiment.

The term “effective amount” as used herein refers to that amount of the(modified) non-natural amino acid polypeptide being administered whichwill relieve to some extent one or more of the symptoms of the disease,condition or disorder being treated. Compositions containing the(modified) non-natural amino acid polypeptide described herein can beadministered for prophylactic, enhancing, and/or therapeutic treatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancingthe effect of therapeutic agents, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents on a system. An “enhancing-effectiveamount,” as used herein, refers to an amount adequate to enhance theeffect of another therapeutic agent in a desired system. When used in apatient, amounts effective for this use will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

The term “modified,” as used herein refers to the presence of apost-translational modification on a polypeptide. The form “(modified)”term means that the polypeptides being discussed are optionallymodified, that is, the polypeptides under discussion can be modified orunmodified.

The term “post-translationally modified” and “modified” refers to anymodification of a natural or non-natural amino acid that occurs to suchan amino acid after it has been incorporated into a polypeptide chain.The term encompasses, by way of example only, co-translational in vivomodifications, post-translational in vivo modifications, andpost-translational in vitro modifications.

In prophylactic applications, compositions containing the (modified)non-natural amino acid polypeptide are administered to a patientsusceptible to or otherwise at risk of a particular disease, disorder orcondition. Such an amount is defined to be a “prophylactically effectiveamount.” In this use, the precise amounts also depend on the patient'sstate of health, weight, and the like. It is considered well within theskill of the art for one to determine such prophylactically effectiveamounts by routine experimentation (e.g., a dose escalation clinicaltrial).

The term “protected” refers to the presence of a “protecting group” ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin or with the methods and compositions described herein.

By way of example only, blocking/protecting groups may be selected from:

Other protecting groups are described in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y.,1999, which is incorporated herein by reference in its entirety.

In therapeutic applications, compositions containing the (modified)non-natural amino acid polypeptide are administered to a patient alreadysuffering from a disease, condition or disorder, in an amount sufficientto cure or at least partially arrest the symptoms of the disease,disorder or condition. Such an amount is defined to be a“therapeutically effective amount,” and will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

The term “treating” is used to refer to either prophylactic and/ortherapeutic treatments.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed.

DETAILED DESCRIPTION

I. Introduction

ABP molecules comprising at least one unnatural amino acid are providedin the invention. In certain embodiments of the invention, ABP with atleast one unnatural amino acid includes at least one post-translationalmodification. In one embodiment, the at least one post-translationalmodification comprises attachment of a molecule including but notlimited to, a label, a dye, a polymer, a water-soluble polymer, aderivative of polyethylene glycol, a photocrosslinker, a cytotoxiccompound, a radionuclide, a drug, an affinity label, a photoaffinitylabel, a reactive compound, a resin, a second protein or polypeptide orpolypeptide analog, an antibody or antibody fragment, a metal chelator,a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, aRNA, an antisense polynucleotide, a water-soluble dendrimer, acyclodextrin, an inhibitory ribonucleic acid, a biomaterial, ananoparticle, a spin label, a fluorophore, a metal-containing moiety, aradioactive moiety, a novel functional group, a group that covalently ornoncovalently interacts with other molecules, a photocaged moiety, aphotoisomerizable moiety, biotin, a derivative of biotin, a biotinanalogue, a moiety incorporating a heavy atom, a chemically cleavablegroup, a photocleavable group, an elongated side chain, a carbon-linkedsugar, a redox-active agent, an amino thioacid, a toxic moiety, anisotopically labeled moiety, a biophysical probe, a phosphorescentgroup, a chemiluminescent group, an electron dense group, a magneticgroup, an intercalating group, a chromophore, an energy transfer agent,a biologically active agent, a detectable label, a small molecule, orany combination of the above or any other desirable compound orsubstance, comprising a second reactive group to at least one unnaturalamino acid comprising a first reactive group utilizing chemistrymethodology that is known to one of ordinary skill in the art to besuitable for the particular reactive groups. For example, the firstreactive group is an alkynyl moiety (including but not limited to, inthe unnatural amino acid p-propargyloxyphenylalanine, where thepropargyl group is also sometimes referred to as an acetylene moiety)and the second reactive group is an azido moiety, and [3+2]cycloadditionchemistry methodologies are utilized. In another example, the firstreactive group is the azido moiety (including but not limited to, in theunnatural amino acid p-azido-L-phenylalanine) and the second reactivegroup is the alkynyl moiety. In certain embodiments of the modified ABPpolypeptide of the present invention, at least one unnatural amino acid(including but not limited to, unnatural amino acid containing a ketofunctional group) comprising at least one post-translationalmodification, is used where the at least one post-translationalmodification comprises a saccharide moiety. In certain embodiments, thepost-translational modification is made in vivo in a eukaryotic cell orin a non-eukaryotic cell.

In certain embodiments, the protein includes at least onepost-translational modification that is made in vivo by one host cell,where the post-translational modification is not normally made byanother host cell type. In certain embodiments, the protein includes atleast one post-translational modification that is made in vivo by aeukaryotic cell, where the post-translational modification is notnormally made by a non-eukaryotic cell. Examples of post-translationalmodifications include, but are not limited to, acetylation, acylation,lipid-modification, palmitoylation, palmitate addition, phosphorylation,glycolipid-linkage modification, and the like. In one embodiment, thepost-translational modification comprises attachment of anoligosaccharide to an asparagine by a GlcNAc-asparagine linkage(including but not limited to, where the oligosaccharide comprises(GlcNAc-Man)₂-Man-GlcNAc-GlcNAc, and the like). In another embodiment,the post-translational modification comprises attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine, a GalNAc-threonine, aGlcNAc-serine, or a GlcNAc-threonine linkage. In certain embodiments, aprotein or polypeptide of the invention can comprise a secretion orlocalization sequence, an epitope tag, a FLAG tag, a polyhistidine tag,a GST fusion, and/or the like. Examples of secretion signal sequencesinclude, but are not limited to, a prokaryotic secretion signalsequence, an eukaryotic secretion signal sequence, an eukaryoticsecretion signal sequence 5′-optimized for bacterial expression, a novelsecretion signal sequence, pectate lyase secretion signal sequence, OmpA secretion signal sequence, and a phage secretion signal sequence.Examples of secretion signal sequences, include, but are not limited to,STII (prokaryotic), Fd GIII and M13 (phage), Bgl2 (yeast), and thesignal sequence bla derived from a transposon.

An antigen-binding polypeptide comprising a non-natural amino acid maybe used to modulate the therapeutic half-life, serum half-life, orcirculation time of biologically active molecules, including but notlimited to, small molecules, peptides, and oligonucleotides. Such smallmolecules, peptides, and oligonucleotides may have biological activitiesthat include, but are not limited to, binding and/or recognition of atarget molecule or cell type, anti-tumor, anti-angiogenic, anti-viral,and apoptotic activities. In addition, the antigen-binding polypeptidecomprising a non-natural amino acid may provide a desired activity,including, but not limited to, effector function such as ADCC,phagocytosis, or complement-dependent cytotoxicity, activation ofprodrugs, enzymatic activity, catalytic activity, blocking ofprotein-protein interactions, binding to a desired antigen, andtargeting of the small molecule to a desired site. The blocking ofprotein-protein interactions of an ABP may modulate one or moreactivities of the attached biologically active molecule. Small moleculesmay be used as antagonists to interfere with the binding activities ofother proteins or molecules.

The antigen-binding polypeptide and the small molecule may be joined bya linker, polymer or covalent bond. The linker, polymer, or smallmolecule itself may comprise a functional group that is unreactivetoward the 20 common amino acids. The linker or polymer may bebifunctional. One or more bonds involved in joining the antigen-bindingpolypeptide via the linker, polymer, or covalent bond to thebiologically active molecule may be irreversible, reversible or labileunder desired conditions. One or more bonds involved in joining theantigen-binding polypeptide via the linker, polymer, or covalent bond toa molecule may allow modulated release of the antigen-bindingpolypeptide or other molecule. A diversity of small molecules may begenerated by one skilled in the art by chemical means, isolation asnatural products, or other means.

Rader et al. in Proc Natl Acad Sci USA. 2003 Apr. 29; 100(9):5396-400,which is incorporated by reference herein, describe a method to provideeffector function and extended serum half-life to small syntheticmolecules via reacting them with a generic antibody molecule. Thecomplex described was created by a reversible covalent bond between mAb38C2, a catalytic antibody that mimics natural aldolase enzymes, and adiketone derivative of an integrin targeting Arg-Gly-Asp peptidomimeticvia a reactive lysine residue on the antibody. In addition to anincrease in half life of the peptidomimetic, the complex showedselective retargeting of the antibody to the surface of integrin α_(v)β₃and α_(v)β₅ expressing cells.

The protein or polypeptide of interest can contain at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or ten or more unnaturalamino acids. The unnatural amino acids can be the same or different, forexample, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentsites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent unnatural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein is substituted with an unnatural aminoacid.

The present invention provides methods and compositions based onantigen-binding polypeptides, or ABP, comprising at least onenon-naturally encoded amino acid. Introduction of at least onenon-naturally encoded amino acid into an ABP can allow for theapplication of conjugation chemistries that involve specific chemicalreactions, including, but not limited to, with one or more non-naturallyencoded amino acids while not reacting with the commonly occurring 20amino acids. In some embodiments, the ABP comprising the non-naturallyencoded amino acid is linked to a water soluble polymer, such aspolyethylene glycol (PEG), via the side chain of the non-naturallyencoded amino acid. This invention provides a highly efficient methodfor the selective modification of proteins with PEG derivatives, whichinvolves the selective incorporation of non-genetically encoded aminoacids, including but not limited to, those amino acids containingfunctional groups or substituents not found in the 20 naturallyincorporated amino acids, including but not limited to a ketone, anazide or acetylene moiety, into proteins in response to a selector codonand the subsequent modification of those amino acids with a suitablyreactive PEG derivative. Once incorporated, the amino acid side chainscan then be modified by utilizing chemistry methodologies known to thoseof ordinary skill in the art to be suitable for the particularfunctional groups or substituents present in the naturally encoded aminoacid. Known chemistry methodologies of a wide variety are suitable foruse in the present invention to incorporate a water soluble polymer intothe protein. Such methodologies include but are not limited to a Huisgen[3+2]cycloaddition reaction (see, e.g., Padwa, A. in ComprehensiveOrganic Synthesis. Vol. 4, (1991) Ed. Trost, B. M., Pergamon, Oxford, p.1069-1109; and, Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry,(1984) Ed. Padwa, A., Wiley, New York, p. 1-176) with, including but notlimited to, acetylene or azide derivatives, respectively.

Because the Huisgen [3+2]cycloaddition method involves a cycloadditionrather than a nucleophilic substitution reaction, proteins can bemodified with extremely high selectivity. The reaction can be carriedout at room temperature in aqueous conditions with excellentregioselectivity (1.4>1.5) by the addition of catalytic amounts of Cu(I)salts to the reaction mixture. See, e.g., Tornoe, et al., (2002) Org.Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int.Ed. 41:2596-2599; and WO 03/101972. A molecule that can be added to aprotein of the invention through a [3+2]cycloaddition includes virtuallyany molecule with a suitable functional group or substituent includingbut not limited to an azido or acetylene derivative. These molecules canbe added to an unnatural amino acid with an acetylene group, includingbut not limited to, p-propargyloxyphenylalanine, or azido group,including but not limited to p-azido-phenylalanine, respectively.

The five-membered ring that results from the Huisgen [3+2]cycloadditionis not generally reversible in reducing environments and is stableagainst hydrolysis for extended periods in aqueous environments.Consequently, the physical and chemical characteristics of a widevariety of substances can be modified under demanding aqueous conditionswith the active PEG derivatives of the present invention. Even moreimportant, because the azide and acetylene moieties are specific for oneanother (and do not, for example, react with any of the 20 common,genetically-encoded amino acids), proteins can be modified in one ormore specific sites with extremely high selectivity.

The invention also provides water soluble and hydrolytically stablederivatives of PEG derivatives and related hydrophilic polymers havingone or more acetylene or azide moieties. The PEG polymer derivativesthat contain acetylene moieties are highly selective for coupling withazide moieties that have been introduced selectively into proteins inresponse to a selector codon. Similarly, PEG polymer derivatives thatcontain azide moieties are highly selective for coupling with acetylenemoieties that have been introduced selectively into proteins in responseto a selector codon.

More specifically, the azide moieties comprise, but are not limited to,alkyl azides, aryl azides and derivatives of these azides. Thederivatives of the alkyl and aryl azides can include other substituentsso long as the acetylene-specific reactivity is maintained. Theacetylene moieties comprise alkyl and aryl acetylenes and derivatives ofeach. The derivatives of the alkyl and aryl acetylenes can include othersubstituents so long as the azide-specific reactivity is maintained.

ABP comprising a non-naturally encoded amino acid may be used in assaysthat utilize the specificity of antibodies. For example, ABP moleculesof the invention may be used to screen a population of potentialantigens.

The present invention provides conjugates of substances having a widevariety of functional groups, substituents or moieties, with othersubstances including but not limited to a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a cytotoxic compound; a radionuclide; a drug; anaffinity label; a photoaffinity label; a reactive compound; a resin; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a water-soluble dendrimer; a cyclodextrin; an inhibitoryribonucleic acid; a biomaterial; a nanoparticle; a spin label; afluorophore, a metal-containing moiety; a radioactive moiety; a novelfunctional group; a group that covalently or noncovalently interactswith other molecules; a photocaged moiety; a photoisomerizable moiety;biotin; a derivative of biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; a small molecule; or anycombination of the above, or any other desirable compound or substance).The present invention also includes conjugates of substances havingazide or acetylene moieties with PEG polymer derivatives having thecorresponding acetylene or azide moieties. For example, a PEG polymercontaining an azide moiety can be coupled to a biologically activemolecule at a position in the protein that contains a non-geneticallyencoded amino acid bearing an acetylene functionality. The linkage bywhich the PEG and the biologically active molecule are coupled includesbut is not limited to the Huisgen [3+2]cycloaddition product.

It is well established in the art that PEG can be used to modify thesurfaces of biomaterials (see, e.g., U.S. Pat. No. 6,610,281; Mehvar,R., J. Pharmaceut. Sci., 3(1):125-136 (2000) which are incorporated byreference herein). The invention also includes biomaterials comprising asurface having one or more reactive azide or acetylene sites and one ormore of the azide- or acetylene-containing polymers of the inventioncoupled to the surface via the Huisgen [3+2]cycloaddition linkage.Biomaterials and other substances can also be coupled to the azide- oracetylene-activated polymer derivatives through a linkage other than theazide or acetylene linkage, such as through a linkage comprising acarboxylic acid, amine, alcohol or thiol moiety, to leave the azide oracetylene moiety available for subsequent reactions.

The invention includes a method of synthesizing the azide- andacetylene-containing polymers of the invention. In the case of theazide-containing PEG derivative, the azide can be bonded directly to acarbon atom of the polymer. Alternatively, the azide-containing PEGderivative can be prepared by attaching a linking agent that has theazide moiety at one terminus to a conventional activated polymer so thatthe resulting polymer has the azide moiety at its terminus. In the caseof the acetylene-containing PEG derivative, the acetylene can be bondeddirectly to a carbon atom of the polymer. Alternatively, theacetylene-containing PEG derivative can be prepared by attaching alinking agent that has the acetylene moiety at one terminus to aconventional activated polymer so that the resulting polymer has theacetylene moiety at its terminus.

More specifically, in the case of the azide-containing PEG derivative, awater soluble polymer having at least one active hydroxyl moietyundergoes a reaction to produce a substituted polymer having a morereactive moiety, such as a mesylate, tresylate, tosylate or halogenleaving group, thereon. The preparation and use of PEG derivativescontaining sulfonyl acid halides, halogen atoms and other leaving groupsare well known to the skilled artisan. The resulting substituted polymerthen undergoes a reaction to substitute for the more reactive moiety anazide moiety at the terminus of the polymer. Alternatively, a watersoluble polymer having at least one active nucleophilic or electrophilicmoiety undergoes a reaction with a linking agent that has an azide atone terminus so that a covalent bond is formed between the PEG polymerand the linking agent and the azide moiety is positioned at the terminusof the polymer. Nucleophilic and electrophilic moieties, includingamines, thiols, hydrazides, hydrazines, alcohols, carboxylates,aldehydes, ketones, thioesters and the like, are well known to theskilled artisan.

More specifically, in the case of the acetylene-containing PEGderivative, a water soluble polymer having at least one active hydroxylmoiety undergoes a reaction to displace a halogen or other activatedleaving group from a precursor that contains an acetylene moiety.Alternatively, a water soluble polymer having at least one activenucleophilic or electrophilic moiety undergoes a reaction with a linkingagent that has an acetylene at one terminus so that a covalent bond isformed between the PEG polymer and the linking agent and the acetylenemoiety is positioned at the terminus of the polymer. The use of halogenmoieties, activated leaving group, nucleophilic and electrophilicmoieties in the context of organic synthesis and the preparation and useof PEG derivatives is well established to practitioners in the art.

The invention also provides a method for the selective modification ofproteins to add other substances to the modified protein, including butnot limited to water soluble polymers such as PEG and PEG derivativescontaining an azide or acetylene moiety. The azide- andacetylene-containing PEG derivatives can be used to modify theproperties of surfaces and molecules where biocompatibility, stability,solubility and lack of immunogenicity are important, while at the sametime providing a more selective means of attaching the PEG derivativesto proteins than was previously known in the art.

II. Antigen-Binding Polypeptides

There is a wide variety of ABP's. ABPs are themselves specific for avery wide variety of antigens. There is also a large number of a verywide variety of ABP fragments that are antigen-specific. ABP thereforeis intended to include any polypeptide that demonstrates an ability tospecifically bind to a target molecule or antigen. Any known antibody orantibody fragment is an ABP.

ABP's of the invention may comprise an Fc region or Fc-like region. TheFc domain provides the link to effector functions such as complement orphagocytic cells. The Fc portion of an immunoglobulin has a long plasmahalf-life, whereas the Fab is short-lived (Capon, et al. (1989), Nature,337:525-531). When constructed together with a therapeutic protein, anFc domain can provide longer half-life or incorporate such functions asFc receptor binding, protein A binding, complement fixation and perhapseven placental transfer. For example, the Fc region of an IgG1 antibodyhas been fused to the N-terminal end of CD30-L, a molecule which bindsCD30 receptors expressed on Hodgkin's Disease tumor cells, anaplasticlymphoma cells, T-cell leukemia cells and other malignant cell types(U.S. Pat. No. 5,480,981). IL-10, an anti-inflammatory and antirejectionagent has been fused to murine Fcγ2a in order to increase the cytokine'sshort circulating half-life. Zheng, X. et al. (1995), The Journal ofImmunology, 154: 5590-5600. Studies have also evaluated the use of tumornecrosis factor receptor linked with the Fc protein of human IgG1 totreat patients with septic shock. Fisher, C. et al., N. Engl. J. Med.,334: 1697-1702 (1996); Van Zee, K. et al., The Journal of Immunology,156: 2221-2230 (1996) and rheumatoid arthritis (Moreland, et al. (1997),N. Engl. J. Med., 337(3):141-147. Fc has also been fused with CD4receptor to produce a therapeutic protein for treatment of AIDS (Caponet al. (1989), Nature, 337:525-531). In addition, the N-terminus ofinterleukin 2 has also been fused to the Fc portion of IgG1 or IgG3 toovercome the short half life of interleukin 2 and its systemic toxicity(Harvill et al. (1995), Immunotechnology, 1: 95-105).

It is well known that Fc regions of antibodies are made up of monomericpolypeptide segments that may be linked into dimeric or multimeric formsby disulfide bonds or by non-covalent association. The number ofintermolecular disulfide bonds between monomeric subunits of native Fcmolecules ranges from 1 to 4 depending on the class (e.g., IgG, IgA,IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2) of antibodyinvolved. The term “Fc” as used herein is generic to the monomeric,dimeric, and multimeric forms of Fc molecules. It should be noted thatFc monomers will spontaneously dimerize when the appropriate Cysresidues are present unless particular conditions are present thatprevent dimerization through disulfide bond formation. Even if the Cysresidues that normally form disulfide bonds in the Fc dimer are removedor replaced by other residues, the monomeric chains will generallydimerize through non-covalent interactions. The term “Fc” herein is usedto mean any of these forms: the native monomer, the native dimer(disulfide bond linked), modified dimers (disulfide and/ornon-covalently linked), and modified monomers (i.e., derivatives).

Variants, analogs or derivatives of the Fc portion may be constructedby, for example, making various substitutions of residues or sequencesincluding non-naturally encoded amino acids. Variant (or analog)polypeptides include insertion variants, wherein one or more amino acidresidues supplement an Fc amino acid sequence. Insertions may be locatedat either or both termini of the protein, or may be positioned withininternal regions of the Fc amino acid sequence. Insertional variantswith additional residues at either or both termini can include forexample, fusion proteins and proteins including amino acid tags orlabels. For example, the Fc molecule may optionally contain anN-terminal Met, especially when the molecule is expressed recombinantlyin a bacterial cell such as E. coli. In Fc deletion variants, one ormore amino acid residues in an Fc polypeptide are removed. Deletions canbe effected at one or both termini of the Fc polypeptide, or withremoval of one or more residues within the Fc amino acid sequence.Deletion variants, therefore, include all fragments of an Fc polypeptidesequence. In Fc substitution variants, one or more amino acid residuesof an Fc polypeptide are removed and replaced with alternative residues.In one aspect, the substitutions are conservative in nature, however,the invention embraces substitutions that are also non-conservative. Forexample, cysteine residues can be deleted or replaced with other aminoacids to prevent formation of some or all disulfide crosslinks of the Fcsequences. A protein may have one or more cysteine residues, and one mayremove each of these cysteine residues or substitute one or more suchcysteine residues with other amino acids, such as Ala or Ser, or anon-naturally encoded amino acid. As another example, modifications mayalso be made to introduce amino acid substitutions to (1) ablate the Fcreceptor binding site; (2) ablate the complement (Clq) binding site;and/or to (3) ablate the antibody dependent cell-mediated cytotoxicity(ADCC) site. Such sites are known in the art, and any knownsubstitutions are within the scope of Fc as used herein. For example,see Molecular Immunology, Vol. 29, No. 5, 633-639 (1992) with regards toADCC sites in IgG1. Likewise, one or more tyrosine residues can bereplaced by phenylalanine residues as well. In addition, other variantamino acid insertions, deletions (e.g., from 1-25 amino acids) and/orsubstitutions are also contemplated and are within the scope of thepresent invention. Conservative amino acid substitutions will generallybe preferred. Furthermore, alterations may be in the form of alteredamino acids, such as peptidomimetics or D-amino acids.

Fc sequences may also be derivatized, i.e., bearing modifications otherthan insertion, deletion, or substitution of amino acid residues.Preferably, the modifications are covalent in nature, and include forexample, chemical bonding with polymers, lipids, other organic moieties,and inorganic moieties. Derivatives of the invention may be prepared toincrease circulating half-life, or may be designed to improve targetingcapacity for the polypeptide to desired cells, tissues, or organs. It isalso possible to use the salvage receptor binding domain of the intactFc molecule as the Fc part of the inventive compounds, such as describedin WO 96/32478, entitled “Altered Polypeptides with IncreasedHalf-Life”. Additional members of the class of molecules designated asFc herein are those that are described in WO 97/34631, entitled“Immunoglobulin-Like Domains with Increased Half-Lives”. Both of thepublished PCT applications cited in this paragraph are herebyincorporated by reference.

Additional ABPs are likely to be discovered in the future. New ABPs canbe identified through computer-aided secondary and tertiary structureanalyses of the predicted protein sequences, and by selection techniquesdesigned to identify molecules that bind to a particular target. Suchlater discovered ABPs also are included within this invention.

Thus, the description of ABPs is provided for illustrative purposes andby way of example only and not as a limit on the scope of the methods,compositions, strategies and techniques described herein. Further,reference to ABP's in this application is intended to use the genericterm as an example of any ABP. Thus, it is understood that themodifications and chemistries described herein with reference to aspecific antigen-binding polypeptide or protein can be equally appliedto any antigen-binding polypeptide, including those specifically listedherein.

III. General Recombinant Nucleic Acid Methods for Use with the Invention

In numerous embodiments of the present invention, nucleic acids encodinga ABP of interest will be isolated, cloned and often altered usingrecombinant methods. Such embodiments are used, including but notlimited to, for protein expression or during the generation of variants,derivatives, expression cassettes, or other sequences derived from anantigen-binding polypeptide. In some embodiments, the sequences encodingthe polypeptides of the invention are operably linked to a heterologouspromoter.

A nucleotide sequence encoding an antigen-binding polypeptide comprisinga non-naturally encoded amino acid may be synthesized on the basis ofthe amino acid sequence of the parent polypeptide and then changing thenucleotide sequence so as to effect introduction (i.e., incorporation orsubstitution) or removal (i.e., deletion or substitution) of therelevant amino acid residue(s). The nucleotide sequence may beconveniently modified by site-directed mutagenesis in accordance withconventional methods. Alternatively, the nucleotide sequence may beprepared by chemical synthesis, including but not limited to, by usingan oligonucleotide synthesizer, wherein oligonucleotides are designedbased on the amino acid sequence of the desired polypeptide, andpreferably selecting those codons that are favored in the host cell inwhich the recombinant polypeptide will be produced. For example, severalsmall oligonucleotides coding for portions of the desired polypeptidemay be synthesized and assembled by PCR, ligation or ligation chainreaction. See, e.g., Barany, et al., Proc. Natl. Acad. Sci. 88: 189-193(1991); U.S. Pat. No. 6,521,427 which are incorporated by referenceherein.

This invention utilizes routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, includingbut not limited to, the generation of genes that include selector codonsfor production of proteins that include unnatural amino acids,orthogonal tRNAs, orthogonal synthetases, and pairs thereof.

Various types of mutagenesis are used in the invention for a variety ofpurposes, including but not limited to, to produce libraries of tRNAs,to produce libraries of synthetases, to produce selector codons, toinsert selector codons that encode unnatural amino acids in a protein orpolypeptide of interest. They include but are not limited tosite-directed, random point mutagenesis, homologous recombination, DNAshuffling or other recursive mutagenesis methods, chimeric construction,mutagenesis using uracil containing templates, oligonucleotide-directedmutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesisusing gapped duplex DNA or the like, or any combination thereof.Additional suitable methods include point mismatch repair, mutagenesisusing repair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,including but not limited to, involving chimeric constructs, are alsoincluded in the present invention. In one embodiment, mutagenesis can beguided by known information of the naturally occurring molecule oraltered or mutated naturally occurring molecule, including but notlimited to, sequence, sequence comparisons, physical properties,secondary, tertiary, or quaternary structure, crystal structure or thelike.

The texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling et al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol.154: 329-350 (1987); Zoller & Smith, Oligonucleotide-directedmutagenesis using M13-derived vectors: an efficient and generalprocedure for the production of point mutations in any DNA fragment,Nucleic Acids Res. 10:6487-6500 (1982); Zoller & Smith,Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors, Methods in Enzymol. 100:468-500 (1983); Zoller & Smith,Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template, Methods inEnzymol. 154:329-350 (1987); Taylor et al., The use ofphosphorothioate-modified DNA in restriction enzyme reactions to preparenicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985); Taylor et al., Therapid generation of oligonucleotide-directed mutations at high frequencyusing phosphorothioate-modified DNA, Nucl. Acids Res. 13: 8765-8787(1985); Nakamaye & Eckstein, Inhibition of restriction endonuclease NciI cleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenesis, Nucl. Acids Res. 14: 9679-9698(1986); Sayers et al., 5′-3′ Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis, Nucl. Acids Res. 16:791-802(1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Different base/base mismatches arecorrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E. coli, Cell 38:879-887 (1984); Carter etal., Improved oligonucleotide site-directed mutagenesis using M13vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Improvedoligonucleotide-directed mutagenesis using M13 vectors, Methods inEnzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use ofoligonucleotides to generate large deletions, Nucl. Acids Res. 14: 5115(1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakmar and Khorana, Total synthesis and expression of a gene forthe alpha-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Grundströmet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001); W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

The invention also relates to eukaryotic host cells, non-eukaryotic hostcells, and organisms for the in vivo incorporation of an unnatural aminoacid via orthogonal tRNA/RS pairs. Host cells are genetically engineered(including but not limited to, transformed, transduced or transfected)with the polynucleotides of the invention or constructs which include apolynucleotide of the invention, including but not limited to, a vectorof the invention, which can be, for example, a cloning vector or anexpression vector. The vector can be, for example, in the form of aplasmid, a bacterium, a virus, a naked polynucleotide, or a conjugatedpolynucleotide. The vectors are introduced into cells and/ormicroorganisms by standard methods including electroporation (From etal., Proc. Natl. Acad. Sci. USA 82, 5824 (1985), infection by viralvectors, high velocity ballistic penetration by small particles with thenucleic acid either within the matrix of small beads or particles, or onthe surface (Klein et al., Nature 327, 70-73 (1987)).

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, including but not limited to for cell isolation and culture(e.g., for subsequent nucleic acid isolation) include Freshney (1994)Culture of Animal Cells. a Manual of Basic Technique, third edition,Wiley-Liss, New York and the references cited therein; Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in the invention. Theseinclude: fusion of the recipient cells with bacterial protoplastscontaining the DNA, electroporation, projectile bombardment, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, a plethoraof kits are commercially available for the purification of plasmids frombacteria, (see, e.g., EasyPrep™, Flexiprep™, both from PharmaciaBiotech; StrataClean™ from Stratagene; and, QIAprep™ from Qiagen). Theisolated and purified plasmids are then further manipulated to produceother plasmids, used to transfect cells or incorporated into relatedvectors to infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (including but not limited to,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith,Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987); Schneider,B., et al., Protein Expr. Purif. 6435:10 (1995); Ausubel, Sambrook,Berger (all supra). A catalogue of bacteria and bacteriophages usefulfor cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue ofBacteria and Bacteriophage (1992) Ghema et al. (eds) published by theATCC. Additional basic procedures for sequencing, cloning and otheraspects of molecular biology and underlying theoretical considerationsare also found in Watson et al. (1992) Recombinant DNA Second EditionScientific American Books, NY. In addition, essentially any nucleic acid(and virtually any labeled nucleic acid, whether standard ornon-standard) can be custom or standard ordered from any of a variety ofcommercial sources, such as the Midland Certified Reagent Company(Midland, Tex. available on the World Wide Web at mcrc.com), The GreatAmerican Gene Company (Ramona, Calif. available on the World Wide Web atgenco.com), ExpressGen Inc. (Chicago, Ill. available on the World WideWeb at expressgen.com), Operon Technologies Inc. (Alameda, Calif.) andmany others.

Selector Codons

Selector codons of the invention expand the genetic codon framework ofprotein biosynthetic machinery. For example, a selector codon includes,but is not limited to, a unique three base codon, a nonsense codon, suchas a stop codon, including but not limited to, an amber codon (UAG), oran opal codon (UGA), an unnatural codon, a four or more base codon, arare codon, or the like. It is readily apparent to those of ordinaryskill in the art that there is a wide range in the number of selectorcodons that can be introduced into a desired gene, including but notlimited to, one or more, two or more, more than three, 4, 5, 6, 7, 8, 9,10 or more in a single polynucleotide encoding at least a portion ofABP.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of unnatural amino acids in vivoin a eukaryotic cell. For example, an O-tRNA is produced that recognizesthe stop codon, including but not limited to, UAG, and is aminoacylatedby an O—RS with a desired unnatural amino acid. This O-tRNA is notrecognized by the naturally occurring host's aminoacyl-tRNA synthetases.Conventional site-directed mutagenesis can be used to introduce the stopcodon, including but not limited to, TAG, at the site of interest in apolypeptide of interest. See, e.g., Sayers, J. R., et al. (1988), 5′,3′Exonuclease in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res. 791-802. When the O—RS, O-tRNA and thenucleic acid that encodes the polypeptide of interest are combined invivo, the unnatural amino acid is incorporated in response to the UAGcodon to give a polypeptide containing the unnatural amino acid at thespecified position.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the eukaryotic host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, including but not limited to, the ambersuppressor tRNA, and a eukaryotic release factor (including but notlimited to, eRF) (which binds to a stop codon and initiates release ofthe growing peptide from the ribosome), the suppression efficiency canbe modulated by, including but not limited to, increasing the expressionlevel of O-tRNA, and/or the suppressor tRNA.

Selector codons also comprise extended codons, including but not limitedto, four or more base codons, such as, four, five, six or more basecodons. Examples of four base codons include, including but not limitedto, AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codonsinclude, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU,UAGGC and the like. A feature of the invention includes using extendedcodons based on frameshift suppression. Four or more base codons caninsert, including but not limited to, one or multiple unnatural aminoacids into the same protein. For example, in the presence of mutatedO-tRNAs, including but not limited to, a special frameshift suppressortRNAs, with anticodon loops, for example, with at least 8-10 ntanticodon loops, the four or more base codon is read as single aminoacid. In other embodiments, the anticodon loops can decode, includingbut not limited to, at least a four-base codon, at least a five-basecodon, or at least a six-base codon or more. Since there are 256possible four-base codons, multiple unnatural amino acids can be encodedin the same cell using a four or more base codon. See, Anderson et al.,(2002) Exploring the Limits of Codon and Anticodon Size, Chemistry andBiology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol. Biol., 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in the present invention,which can reduce missense readthrough and frameshift suppression atother unwanted sites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology, 20:177-182. Other relevantpublications are listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11586; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274. A3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc. 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., Tae et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is incorporated into a gene but isnot translated into protein. The sequence contains a structure thatserves as a cue to induce the ribosome to hop over the sequence andresume translation downstream of the insertion.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods well-known to one of skill in the art and described hereinto include, for example, one or more selector codon for theincorporation of an unnatural amino acid. For example, a nucleic acidfor a protein of interest is mutagenized to include one or more selectorcodon, providing for the incorporation of one or more unnatural aminoacids. The invention includes any such variant, including but notlimited to, mutant, versions of any protein, for example, including atleast one unnatural amino acid. Similarly, the invention also includescorresponding nucleic acids, i.e., any nucleic acid with one or moreselector codon that encodes one or more unnatural amino acid.

Nucleic acid molecules encoding a protein of interest such as ABP may bereadily mutated to introduce a cysteine at any desired position of thepolypeptide. Cysteine is widely used to introduce reactive molecules,water soluble polymers, proteins, or a wide variety of other molecules,onto a protein of interest. Methods suitable for the incorporation ofcysteine into a desired position of the antigen-binding polypeptide arewell known in the art, such as those described in U.S. Pat. No.6,608,183, which is incorporated by reference herein, and standardmutagenesis techniques.

IV. Non-Naturally Encoded Amino Acids

A very wide variety of non-naturally encoded amino acids are suitablefor use in the present invention. Any number of non-naturally encodedamino acids can be introduced into ABP. In general, the introducednon-naturally encoded amino acids are substantially chemically inerttoward the 20 common, genetically-encoded amino acids (i.e., alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline). In some embodiments, the non-naturally encoded amino acidsinclude side chain functional groups that react efficiently andselectively with functional groups not found in the 20 common aminoacids (including but not limited to, azido, ketone, aldehyde andaminooxy groups) to form stable conjugates. For example, antigen-bindingpolypeptide that includes a non-naturally encoded amino acid containingan azido functional group can be reacted with a polymer (including butnot limited to, poly(ethylene glycol) or, alternatively, a secondpolypeptide containing an alkyne moiety to form a stable conjugateresulting for the selective reaction of the azide and the alkynefunctional groups to form a Huisgen [3+2]cycloaddition product.

The generic structure of an alpha-amino acid is illustrated as follows(Formula I):

A non-naturally encoded amino acid is typically any structure having theabove-listed formula wherein the R group is any substituent other thanone used in the twenty natural amino acids, and may be suitable for usein the present invention. Because the non-naturally encoded amino acidsof the invention typically differ from the natural amino acids only inthe structure of the side chain, the non-naturally encoded amino acidsform amide bonds with other amino acids, including but not limited to,natural or non-naturally encoded, in the same manner in which they areformed in naturally occurring polypeptides. However, the non-naturallyencoded amino acids have side chain groups that distinguish them fromthe natural amino acids. For example, R optionally comprises an alkyl-,aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-,hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, amino group, or the like orany combination thereof. Other non-naturally occurring amino acids ofinterest that may be suitable for use in the present invention include,but are not limited to, amino acids comprising a photoactivatablecross-linker, spin-labeled amino acids, fluorescent amino acids, metalbinding amino acids, metal-containing amino acids, radioactive aminoacids, amino acids with novel functional groups, amino acids thatcovalently or noncovalently interact with other molecules, photocagedand/or photoisomerizable amino acids, amino acids comprising biotin or abiotin analogue, glycosylated amino acids such as a sugar substitutedserine, other carbohydrate modified amino acids, keto-containing aminoacids, amino acids comprising polyethylene glycol or polyether, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, including but not limited to, polyethers or longchain hydrocarbons, including but not limited to, greater than about 5or greater than about 10 carbons, carbon-linked sugar-containing aminoacids, redox-active amino acids, amino thioacid containing amino acids,and amino acids comprising one or more toxic moiety.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present invention and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Many of the non-naturally encoded amino acids provided herein arecommercially available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA),Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), orPeptech (Burlington, Mass., USA). Those that are not commerciallyavailable are optionally synthesized as provided herein or usingstandard methods known to those of skill in the art. For organicsynthesis techniques, see, e.g., Organic Chemistry by Fessendon andFessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.);Advanced Organic Chemistry by March (Third Edition, 1985, Wiley andSons, New York); and Advanced Organic Chemistry by Carey and Sundberg(Third Edition, Parts A and B, 1990, Plenum Press, New York). See, also,U.S. Patent Application Publications 2003/0082575 and 2003/0108885,which is incorporated by reference herein. In addition to unnaturalamino acids that contain novel side chains, unnatural amino acids thatmay be suitable for use in the present invention also optionallycomprise modified backbone structures, including but not limited to, asillustrated by the structures of Formula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates,including but not limited to, with side chains corresponding to thecommon twenty natural amino acids or unnatural side chains. In addition,substitutions at the α-carbon optionally include, but are not limitedto, L, D, or α-α-disubstituted amino acids such as D-glutamate,D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Otherstructural alternatives include cyclic amino acids, such as prolineanalogues as well as 3, 4, 6, 7, 8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

Many unnatural amino acids are based on natural amino acids, such astyrosine, glutamine, phenylalanine, and the like, and are suitable foruse in the present invention. Tyrosine analogs include, but are notlimited to, para-substituted tyrosines, ortho-substituted tyrosines, andmeta substituted tyrosines, where the substituted tyrosine comprises,including but not limited to, a keto group (including but not limitedto, an acetyl group), a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, an isopropyl group, amethyl group, a C₆-C₂₀ straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, an alkynyl group or the like. In addition,multiply substituted aryl rings are also contemplated. Glutamine analogsthat may be suitable for use in the present invention include, but arenot limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclicderivatives, and amide substituted glutamine derivatives. Examplephenylalanine analogs that may be suitable for use in the presentinvention include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, where the substituent comprises, including but notlimited to, a hydroxy group, a methoxy group, a methyl group, an allylgroup, an aldehyde, an azido, an iodo, a bromo, a keto group (includingbut not limited to, an acetyl group), a benzoyl, an alkynyl group, orthe like. Specific examples of unnatural amino acids that may besuitable for use in the present invention include, but are not limitedto, a p-acetyl-L-phenylalanine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike. Examples of structures of a variety of unnatural amino acids thatmay be suitable for use in the present invention are provided in, forexample, WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids.” See also Kiick et al., (2002) Incorporation of azides intorecombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99:19-24, for additional methionine analogs.

In one embodiment, compositions of ABP that include an unnatural aminoacid (such as p-(propargyloxy)-phenyalanine) are provided. Variouscompositions comprising p-(propargyloxy)-phenyalanine and, including butnot limited to, proteins and/or cells, are also provided. In one aspect,a composition that includes the p-(propargyloxy)-phenyalanine unnaturalamino acid, further includes an orthogonal tRNA. The unnatural aminoacid can be bonded (including but not limited to, covalently) to theorthogonal tRNA, including but not limited to, covalently bonded to theorthogonal tRNA though an amino-acyl bond, covalently bonded to a 3′OHor a 2′OH of a terminal ribose sugar of the orthogonal tRNA, etc.

The chemical moieties via unnatural amino acids that can be incorporatedinto proteins offer a variety of advantages and manipulations of theprotein. For example, the unique reactivity of a keto functional groupallows selective modification of proteins with any of a number ofhydrazine- or hydroxylamine-containing reagents in vitro and in vivo. Aheavy atom unnatural amino acid, for example, can be useful for phasingX-ray structure data. The site-specific introduction of heavy atomsusing unnatural amino acids also provides selectivity and flexibility inchoosing positions for heavy atoms. Photoreactive unnatural amino acids(including but not limited to, amino acids with benzophenone andarylazides (including but not limited to, phenylazide) side chains), forexample, allow for efficient in vivo and in vitro photocrosslinking ofprotein. Examples of photoreactive unnatural amino acids include, butare not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine.The protein with the photoreactive unnatural amino acids can then becrosslinked at will by excitation of the photoreactive group-providingtemporal control. In one example, the methyl group of an unnatural aminocan be substituted with an isotopically labeled, including but notlimited to, methyl group, as a probe of local structure and dynamics,including but not limited to, with the use of nuclear magnetic resonanceand vibrational spectroscopy. Alkynyl or azido functional groups, forexample, allow the selective modification of proteins with moleculesthrough a [3+2]cycloaddition reaction.

A non-natural amino acid incorporated into a polypeptide at the aminoterminus can be composed of an R group that is any substituent otherthan one used in the twenty natural amino acids and a 2^(nd) reactivegroup different from the NH₂ group normally present in α-amino acids(see Formula I). A similar non-natural amino acid can be incorporated atthe carboxyl terminus with a 2^(nd) reactive group different from theCOOH group normally present in α-amino acids (see Formula I).

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids suitable for use in the presentinvention are commercially available, e.g., from Sigma (USA) or Aldrich(Milwaukee, Wis., USA). Those that are not commercially available areoptionally synthesized as provided herein or as provided in variouspublications or using standard methods known to those of skill in theart. For organic synthesis techniques, see, e.g., Organic Chemistry byFessendon and Fessendon, (1982, Second Edition, Willard Grant Press,Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985,Wiley and Sons, New York); and Advanced Organic Chemistry by Carey andSundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York).Additional publications describing the synthesis of unnatural aminoacids include, e.g., WO 2002/085923 entitled “In vivo incorporation ofUnnatural Amino Acids;” Matsoukas et al., (1995) J. Med. Chem., 38,4660-4669; King, F. E. & Kidd, D. A. A. (1949) A New Synthesis ofGlutamine and of γ-Dipeptides of Glutamic Acid from PhthylatedIntermediates. J. Chem. Soc., 3315-3319; Friedman, O. M. & Chatterrji,R. (1959) Synthesis of Derivatives of Glutamine as Model Substrates forAnti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752; Craig, J. C. et al.(1988) Absolute Configuration of the Enantiomers of 7-Chloro-4[[4-(diethylamino)-1-methylbutyl]amino]quinoline (Chloroquine). J. Org.Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. & Frappier, F. (1991)Glutamine analogues as Potential Antimalarials., Eur. J. Med. Chem. 26,201-5; Koskinen, A. M. P. & Rapoport, H. (1989) Synthesis of4-Substituted Prolines as Conformationally Constrained Amino AcidAnalogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. & Rapoport, H.(1985) Synthesis of Optically Pure Pipecolates from L-Asparagine.Application to the Total Synthesis of (+)-Apovincamine through AminoAcid Decarbonylation and Iminium Ion Cyclization. J. Org. Chem.1989:1859-1866; Barton et al., (1987) Synthesis of Novel a-Amino-Acidsand Derivatives Using Radical Chemistry: Synthesis of L- andD-a-Amino-Adipic Acids, L-a-aminopimelic Acid and AppropriateUnsaturated Derivatives. Tetrahedron Lett. 43:4297-4308; and, Subasingheet al., (1992) Quisqualic acid analogues: synthesis of beta-heterocyclic2-aminopropanoic acid derivatives and their activity at a novelquisqualate-sensitized site. J. Med. Chem. 35:4602-7. See also, patentapplications entitled “Protein Arrays,” filed Dec. 22, 2003, Ser. No.10/744,899 and Ser. No. 60/435,821 filed on Dec. 22, 2002.

A. Carbonyl Reactive Groups

Amino acids with a carbonyl reactive group allow for a variety ofreactions to link molecules (including but not limited to, PEG or otherwater soluble molecules) via nucleophilic addition or aldol condensationreactions among others.

Exemplary carbonyl-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group. In some embodiments, n is 1,R₁ is phenyl and R₂ is a simple alkyl (i.e., methyl, ethyl, or propyl)and the ketone moiety is positioned in the para position relative to thealkyl side chain. In some embodiments, n is 1, R₁ is phenyl and R₂ is asimple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety ispositioned in the meta position relative to the alkyl side chain.

The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), which is incorporated by referenceherein. Other carbonyl-containing amino acids can be similarly preparedby one skilled in the art.

In some embodiments, a polypeptide comprising a non-naturally encodedamino acid is chemically modified to generate a reactive carbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-naturally encoded amino acid bearingadjacent hydroxyl and amino groups can be incorporated into thepolypeptide as a “masked” aldehyde functionality. For example,5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.Reaction conditions for generating the aldehyde typically involveaddition of molar excess of sodium metaperiodate under mild conditionsto avoid oxidation at other sites within the polypeptide. The pH of theoxidation reaction is typically about 7.0. A typical reaction involvesthe addition of about 1.5 molar excess of sodium meta periodate to abuffered solution of the polypeptide, followed by incubation for about10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685, which isincorporated by reference herein.

The carbonyl functionality can be reacted selectively with a hydrazine-,hydrazide-, hydroxylamine-, or semicarbazide-containing reagent undermild conditions in aqueous solution to form the corresponding hydrazone,oxime, or semicarbazone linkages, respectively, that are stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonylgroup allows for selective modification in the presence of the otheramino acid side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem.Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug.Chem. 3:138-146 (1992); Mahal, L. K., et al., Science 276:1125-1128(1997).

B. Hydrazine, Hydrazide or Semicarbazide Reactive Groups

Non-naturally encoded amino acids containing a nucleophilic group, suchas a hydrazine, hydrazide or semicarbazide, allow for reaction with avariety of electrophilic groups to form conjugates (including but notlimited to, with PEG or other water soluble polymers).

Exemplary hydrazine, hydrazide or semicarbazide-containing amino acidscan be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X, is O, N, or S or not present; R₂ isH, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, n is 4, R₁ is not present, and X is N. In someembodiments, n is 2, R₁ is not present, and X is not present. In someembodiments, n is 1, R₁ is phenyl, X is O, and the oxygen atom ispositioned para to the aliphatic group on the aryl ring.

Hydrazide-, hydrazine-, and semicarbazide-containing amino acids areavailable from commercial sources. For instance, L-glutamate-γ-hydrazideis available from Sigma Chemical (St. Louis, Mo.). Other amino acids notavailable commercially can be prepared by one skilled in the art. See,e.g., U.S. Pat. No. 6,281,211, which is incorporated by referenceherein.

Polypeptides containing non-naturally encoded amino acids that bearhydrazide, hydrazine or semicarbazide functionalities can be reactedefficiently and selectively with a variety of molecules that containaldehydes or other functional groups with similar chemical reactivity.See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995).The unique reactivity of hydrazide, hydrazine and semicarbazidefunctional groups makes them significantly more reactive towardaldehydes, ketones and other electrophilic groups as compared to thenucleophilic groups present on the 20 common amino acids (including butnot limited to, the hydroxyl group of serine or threonine or the aminogroups of lysine and the N-terminus).

C. Aminooxy-Containing Amino Acids

Non-naturally encoded amino acids containing an aminooxy (also called ahydroxylamine) group allow for reaction with a variety of electrophilicgroups to form conjugates (including but not limited to, with PEG orother water soluble polymers). Like hydrazines, hydrazides andsemicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001). Whereas the result of reaction with a hydrazine group isthe corresponding hydrazone, however, an oxime results generally fromthe reaction of an aminooxy group with a carbonyl-containing group suchas a ketone.

Exemplary amino acids containing aminooxy groups can be represented asfollows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10;Y═C(O) or not present; R₂ is H, an amino acid, a polypeptide, or anamino terminus modification group, and R₃ is H, an amino acid, apolypeptide, or a carboxy terminus modification group. In someembodiments, n is 1, R₁ is phenyl, X is O, m is 1, and Y is present. Insome embodiments, n is 2, R₁ and X are not present, m is 0, and Y is notpresent.

Aminooxy-containing amino acids can be prepared from readily availableamino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R. Brown, J. Org. Chem. 68: 8853-8858 (2003). Certainaminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyricacid), have been isolated from natural sources (Rosenthal, G. et al.,Life Sci. 60: 1635-1641 (1997). Other aminooxy-containing amino acidscan be prepared by one skilled in the art.

D. Azide and Alkyne Reactive Groups

The unique reactivity of azide and alkyne functional groups makes themextremely useful for the selective modification of polypeptides andother biological molecules. Organic azides, particularly aliphaticazides, and alkynes are generally stable toward common reactive chemicalconditions. In particular, both the azide and the alkyne functionalgroups are inert toward the side chains (i.e., R groups) of the 20common amino acids found in naturally-occurring polypeptides. Whenbrought into close proximity, however, the “spring-loaded” nature of theazide and alkyne groups is revealed and they react selectively andefficiently via Huisgen [3+2]cycloaddition reaction to generate thecorresponding triazole. See, e.g., Chin J., et al., Science 301:964-7(2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin,J. W., et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

Because the Huisgen cycloaddition reaction involves a selectivecycloaddition reaction (see, e.g., Padwa, A., in COMPREHENSIVE ORGANICSYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R.in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176)rather than a nucleophilic substitution, the incorporation ofnon-naturally encoded amino acids bearing azide and alkyne-containingside chains permits the resultant polypeptides to be modifiedselectively at the position of the non-naturally encoded amino acid.Cycloaddition reaction involving azide or alkyne-containing ABP can becarried out at room temperature under aqueous conditions by the additionof Cu(II) (including but not limited to, in the form of a catalyticamount of CuSO₄) in the presence of a reducing agent for reducing Cu(II)to Cu(I), in situ, in catalytic amount. See, e.g., Wang, Q., et al., J.Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe, C. W., et al., J. Org.Chem. 67:3057-3064 (2002); Rostovtsev, et al., Angew. Chem. Int. Ed.41:2596-2599 (2002). Exemplary reducing agents include, including butnot limited to, ascorbate, metallic copper, quinine, hydroquinone,vitamin K, glutathione, cysteine, Fe²⁺, Co²⁺, and an applied electricpotential.

In some cases, where a Huisgen [3+2]cycloaddition reaction between anazide and an alkyne is desired, the antigen-binding polypeptidecomprises a non-naturally encoded amino acid comprising an alkyne moietyand the water soluble polymer to be attached to the amino acid comprisesan azide moiety. Alternatively, the converse reaction (i.e., with theazide moiety on the amino acid and the alkyne moiety present on thewater soluble polymer) can also be performed.

The azide functional group can also be reacted selectively with a watersoluble polymer containing an aryl ester and appropriatelyfunctionalized with an aryl phosphine moiety to generate an amidelinkage. The aryl phosphine group reduces the azide in situ and theresulting amine then reacts efficiently with a proximal ester linkage togenerate the corresponding amide. See, e.g., E. Saxon and C. Bertozzi,Science 287, 2007-2010 (2000). The azide-containing amino acid can beeither an alkyl azide (including but not limited to,2-amino-6-azido-1-hexanoic acid) or an aryl azide(p-azido-phenylalanine).

Exemplary water soluble polymers containing an aryl ester and aphosphine moiety can be represented as follows:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

The azide functional group can also be reacted selectively with a watersoluble polymer containing a thioester and appropriately functionalizedwith an aryl phosphine moiety to generate an amide linkage. The arylphosphine group reduces the azide in situ and the resulting amine thenreacts efficiently with the thioester linkage to generate thecorresponding amide. Exemplary water soluble polymers containing athioester and a phosphine moiety can be represented as follows:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

Exemplary alkyne-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10,R₂ is H, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the acetylene moiety is positioned in the paraposition relative to the alkyl side chain. In some embodiments, n is 1,R₁ is phenyl, X is O, m is 1 and the propargyloxy group is positioned inthe para position relative to the alkyl side chain (i.e.,O-propargyl-tyrosine). In some embodiments, n is 1, R₁ and X are notpresent and m is 0 (i.e., proparylglycine).

Alkyne-containing amino acids are commercially available. For example,propargylglycine is commercially available from Peptech (Burlington,Mass.). Alternatively, alkyne-containing amino acids can be preparedaccording to standard methods. For instance, p-propargyloxyphenylalaninecan be synthesized, for example, as described in Deiters, A., et al., J.Am. Chem. Soc. 125: 11782-11783 (2003), and 4-alkynyl-L-phenylalaninecan be synthesized as described in Kayser, B., et al., Tetrahedron53(7): 2475-2484 (1997). Other alkyne-containing amino acids can beprepared by one skilled in the art.

Exemplary azide-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the azide moiety is positioned para to the alkylside chain. In some embodiments, n is 0-4 and R₁ and X are not present,and m=0. In some embodiments, n is 1, R₁ is phenyl, X is O, m is 2 andthe β-azidoethoxy moiety is positioned in the para position relative tothe alkyl side chain.

Azide-containing amino acids are available from commercial sources. Forinstance, 4-azidophenylalanine can be obtained from Chem-ImpexInternational, Inc. (Wood Dale, Ill.). For those azide-containing aminoacids that are not commercially available, the azide group can beprepared relatively readily using standard methods known to those ofskill in the art, including but not limited to, via displacement of asuitable leaving group (including but not limited to, halide, mesylate,tosylate) or via opening of a suitably protected lactone. See, e.g.,Advanced Organic Chemistry by March (Third Edition, 1985, Wiley andSons, New York).

E. Aminothiol Reactive Groups

The unique reactivity of beta-substituted aminothiol functional groupsmakes them extremely useful for the selective modification ofpolypeptides and other biological molecules that contain aldehyde groupsvia formation of the thiazolidine. See, e.g., J. Shao and J. Tam, J. Am.Chem. Soc. 1995, 117 (14) 3893-3899. In some embodiments,beta-substituted aminothiol amino acids can be incorporated into ABPpolypeptides and then reacted with water soluble polymers comprising analdehyde functionality. In some embodiments, a water soluble polymer,drug conjugate or other payload can be coupled to a ABP polypeptidecomprising a beta-substituted aminothiol amino acid via formation of thethiazolidine.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a eukaryotic cell is one issue that istypically considered when designing and selecting unnatural amino acids,including but not limited to, for incorporation into a protein. Forexample, the high charge density of α-amino acids suggests that thesecompounds are unlikely to be cell permeable. Natural amino acids aretaken up into the eukaryotic cell via a collection of protein-basedtransport systems. A rapid screen can be done which assesses whichunnatural amino acids, if any, are taken up by cells. See, e.g., thetoxicity assays in, e.g., the applications entitled “Protein Arrays,”filed Dec. 22, 2003, Ser. No. 10/744,899 and Ser. No. 60/435,821 filedon Dec. 22, 2002; and Liu, D. R. & Schultz, P. G. (1999) Progress towardthe evolution of an organism with an expanded genetic code. PNAS UnitedStates 96:4780-4785. Although uptake is easily analyzed with variousassays, an alternative to designing unnatural amino acids that areamenable to cellular uptake pathways is to provide biosynthetic pathwaysto create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, including butnot limited to, in a eukaryotic cell, the invention provides suchmethods. For example, biosynthetic pathways for unnatural amino acidsare optionally generated in host cell by adding new enzymes or modifyingexisting host cell pathways. Additional new enzymes are optionallynaturally occurring enzymes or artificially evolved enzymes. Forexample, the biosynthesis of p-aminophenylalanine (as presented in anexample in WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids”) relies on the addition of a combination of known enzymesfrom other organisms. The genes for these enzymes can be introduced intoa eukaryotic cell by transforming the cell with a plasmid comprising thegenes. The genes, when expressed in the cell, provide an enzymaticpathway to synthesize the desired compound. Examples of the types ofenzymes that are optionally added are provided in the examples below.Additional enzymes sequences are found, for example, in Genbank.Artificially evolved enzymes are also optionally added into a cell inthe same manner. In this manner, the cellular machinery and resources ofa cell are manipulated to produce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, including but not limited to, asdeveloped by Maxygen, Inc. (available on the World Wide Web atmaxygen.com), is optionally used to develop novel enzymes and pathways.See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNAshuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751.Similarly DesignPath™, developed by Genencor (available on the WorldWide Web at genencor.com) is optionally used for metabolic pathwayengineering, including but not limited to, to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to, identified through functional genomics,and molecular evolution and design. Diversa Corporation (available onthe World Wide Web at diversa.com) also provides technology for rapidlyscreening libraries of genes and gene pathways, including but notlimited to, to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the invention is produced in a concentrationsufficient for efficient protein biosynthesis, including but not limitedto, a natural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a cell is transformed with a plasmid comprisingthe genes used to produce enzymes desired for a specific pathway and anunnatural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the unnatural amino acid forboth ribosomal protein synthesis and cell growth.

Polypeptides with Unnatural Amino Acids

The incorporation of an unnatural amino acid can be done for a varietyof purposes, including but not limited to, tailoring changes in proteinstructure and/or function, changing size, acidity, nucleophilicity,hydrogen bonding, hydrophobicity, accessibility of protease targetsites, targeting to a moiety (including but not limited to, for aprotein array), adding a biologically active molecule, attaching apolymer, attaching a radionuclide, modulating serum half-life,modulating tissue penetration (e.g. tumors), modulating activetransport, modulating tissue, cell or organ specificity, modulatingimmunogenicity, modulating protease resistance, etc. Proteins thatinclude an unnatural amino acid can have enhanced or even entirely newcatalytic or biophysical properties. For example, the followingproperties are optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic ability, half-life (including but not limited to, serumhalf-life), ability to react with other molecules, including but notlimited to, covalently or noncovalently, and the like. The compositionsincluding proteins that include at least one unnatural amino acid areuseful for, including but not limited to, novel therapeutics,diagnostics, catalytic enzymes, industrial enzymes, binding proteins(including but not limited to, antibodies), and including but notlimited to, the study of protein structure and function. See, e.g.,Dougherty, (2000) Unnatural Amino Acids as Probes of protein Structureand Function, Current Opinion in Chemical Biology, 4:645-652.

In one aspect of the invention, a composition includes at least oneprotein with at least one, including but not limited to, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, or at least ten or more unnaturalamino acids. The unnatural amino acids can be the same or different,including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more different sites in the protein that comprise 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more different unnatural amino acids. In anotheraspect, a composition includes a protein with at least one, but fewerthan all, of a particular amino acid present in the protein issubstituted with the unnatural amino acid. For a given protein with morethan one unnatural amino acids, the unnatural amino acids can beidentical or different (including but not limited to, the protein caninclude two or more different types of unnatural amino acids, or caninclude two of the same unnatural amino acid). For a given protein withmore than two unnatural amino acids, the unnatural amino acids can bethe same, different or a combination of a multiple unnatural amino acidof the same kind with at least one different unnatural amino acid.

ABP's of interest with at least one unnatural amino acid are a featureof the invention. The invention also includes polypeptides or proteinswith at least one unnatural amino acid produced using the compositionsand methods of the invention. An excipient (including but not limitedto, a pharmaceutically acceptable excipient) can also be present withthe protein.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in eukaryotic cells, proteins or polypeptides willtypically include eukaryotic post-translational modifications. Incertain embodiments, a protein includes at least one unnatural aminoacid and at least one post-translational modification that is made invivo by a eukaryotic cell, where the post-translational modification isnot made by a prokaryotic cell. For example, the post-translationmodification includes, including but not limited to, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, glycolipid-linkage modification, glycosylation, and thelike. In one aspect, the post-translational modification includesattachment of an oligosaccharide (including but not limited to,(GlcNAc-Man)₂-Man-GlcNAc-GlcNAc)) to an asparagine by aGlcNAc-asparagine linkage. See Table 1 which lists some examples ofN-linked oligosaccharides of eukaryotic proteins (additional residuescan also be present, which are not shown). In another aspect, thepost-translational modification includes attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine or GalNAc-threoninelinkage, or a GlcNAc-serine or a GlcNAc-threonine linkage. TABLE 1EXAMPLES OF OLIGOSACCHARIDES THROUGH GlcNAc-LINKAGE Type Base StructureHigh- mannose

Hybrid

Complex

Xylose

In yet another aspect, the post-translation modification includesproteolytic processing of precursors (including but not limited to,calcitonin precursor, calcitonin gene-related peptide precursor,preproparathyroid hormone, preproinsulin, proinsulin,prepro-opiomelanocortin, pro-opiomelanocortin and the like), assemblyinto a multisubunit protein or macromolecular assembly, translation toanother site in the cell (including but not limited to, to organelles,such as the endoplasmic reticulum, the Golgi apparatus, the nucleus,lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., orthrough the secretory pathway). In certain embodiments, the proteincomprises a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, or the like.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic or non-eukaryoticcell, or in vitro. Thus, in certain embodiments, the post-translationalmodification is through the unnatural amino acid. For example, thepost-translational modification can be through anucleophilic-electrophilic reaction. Most reactions currently used forthe selective modification of proteins involve covalent bond formationbetween nucleophilic and electrophilic reaction partners, including butnot limited to the reaction of α-haloketones with histidine or cysteineside chains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein. In proteinsof the invention, other more selective reactions can be used such as thereaction of an unnatural keto-amino acid with hydrazides or aminooxycompounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) Am.Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science, 276:1125-1128;Wang, et al., (2001) Science 292:498-500; Chin, et al., (2002) Am. Chem.Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl. Acad. Sci.,99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci., 100:56-61;Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin, et al.,(2003) Science, in press. This allows the selective labeling ofvirtually any protein with a host of reagents including fluorophores,crosslinking agents, saccharide derivatives and cytotoxic molecules. Seealso, U.S. patent application Ser. No. 10/686,944 entitled “Glycoproteinsynthesis” filed Oct. 15, 2003 based on U.S. provisional patentapplication Ser. No. 60/419,265, filed Oct. 16, 2002, U.S. provisionalpatent application Ser. No. 60/420,990, filed Oct. 23, 2002, and U.S.provisional patent application Ser. No. 60/441,450, filed Jan. 16, 2003,which are incorporated by reference herein. Post-translationalmodifications, including but not limited to, through an azido aminoacid, can also made through the Staudinger ligation (including but notlimited to, with triarylphosphine reagents). See, e.g., Kiick et al.,(2002) Incorporation of azides into recombinant proteins forchemoselective modification by the Staudinger ligation, PNAS 99:19-24.

This invention provides another highly efficient method for theselective modification of proteins, which involves the geneticincorporation of unnatural amino acids, including but not limited to,containing an azide or alkynyl moiety into proteins in response to aselector codon. These amino acid side chains can then be modified by,including but not limited to, a Huisgen [3+2]cycloaddition reaction(see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991)Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, NewYork, p. 1-176) with, including but not limited to, alkynyl or azidederivatives, respectively. Because this method involves a cycloadditionrather than a nucleophilic substitution, proteins can be modified withextremely high selectivity. This reaction can be carried out at roomtemperature in aqueous conditions with excellent regioselectivity(1.4>1.5) by the addition of catalytic amounts of Cu(I) salts to thereaction mixture. See, e.g., Tornoe, et al., (2002) Org. Chem.67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed.41:2596-2599. Another method that can be used is the ligand exchange ona bisarsenic compound with a tetracysteine motif, see, e.g., Griffin, etal., (1998) Science 281:269-272.

A molecule that can be added to a protein of the invention through a[3+2]cycloaddition includes virtually any molecule with an azide oralkynyl derivative. Molecules include, but are not limited to, dyes,fluorophores, crosslinking agents, saccharide derivatives, polymers(including but not limited to, derivatives of polyethylene glycol),photocrosslinkers, cytotoxic compounds, affinity labels, derivatives ofbiotin, resins, beads, a second protein or polypeptide (or more),polynucleotide(s) (including but not limited to, DNA, RNA, etc.), metalchelators, cofactors, fatty acids, carbohydrates, and the like. Thesemolecules can be added to an unnatural amino acid with an alkynyl group,including but not limited to, p-propargyloxyphenylalanine, or azidogroup, including but not limited to, p-azido-phenylalanine,respectively.

V. In Vivo Generation of ABP Comprising Non-Genetically-Encoded AminoAcids

The antigen-binding polypeptides of the invention can be generated invivo using modified tRNA and tRNA synthetases to add to or substituteamino acids that are not encoded in naturally-occurring systems.

Methods for generating tRNAs and tRNA synthetases which use amino acidsthat are not encoded in naturally-occurring systems are described in,e.g., U.S. Patent Application Publications 2003/0082575 (Ser. No.10/126,927) and 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference herein. These methods involve generating atranslational machinery that functions independently of the synthetasesand tRNAs endogenous to the translation system (and are thereforesometimes referred to as “orthogonal”). Typically, the translationsystem comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyltRNA synthetase (O—RS). Typically, the O—RS preferentially aminoacylatesthe O-tRNA with at least one non-naturally occurring amino acid in thetranslation system and the O-tRNA recognizes at least one selector codonthat is not recognized by other tRNAs in the system. The translationsystem thus inserts the non-naturally-encoded amino acid into a proteinproduced in the system, in response to an encoded selector codon,thereby “substituting” an amino acid into a position in the encodedpolypeptide.

A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases havebeen described in the art for inserting particular synthetic amino acidsinto polypeptides, and are generally suitable for use in the presentinvention. For example, keto-specific O-tRNA/aminoacyl-tRNA synthetasesare described in Wang, L., et al., Proc. Natl. Acad. Sci. USA 100:56-61(2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). ExemplaryO—RS, or portions thereof, are encoded by polynucleotide sequences andinclude amino acid sequences disclosed in U.S. Patent ApplicationPublications 2003/0082575 and 2003/0108885, each incorporated herein byreference. Corresponding O-tRNA molecules for use with the O—RSs arealso described in U.S. Patent Application Publications 2003/0082575(Ser. No. 10/126,927) and 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference herein.

An example of an azide-specific O-tRNA/aminoacyl-tRNA synthetase systemis described in Chin, J. W., et al., J. Am. Chem. Soc. 124:9026-9027(2002). Exemplary O—RS sequences for p-azido-L-Phe include, but are notlimited to, nucleotide sequences SEQ ID NOs: 14-16 and 29-32 and aminoacid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Exemplary O-tRNA sequences suitablefor use in the present invention include, but are not limited to,nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Other examples ofO-tRNA/aminoacyl-tRNA synthetase pairs specific to particularnon-naturally encoded amino acids are described in U.S. PatentApplication Publication 2003/0082575 (Ser. No. 10/126,927) which isincorporated by reference herein. O—RS and O-tRNA that incorporate bothketo- and azide-containing amino acids in S. cerevisiae are described inChin, J. W., et al., Science 301:964-967 (2003).

Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of aspecific codon which encodes the non-naturally encoded amino acid. Whileany codon can be used, it is generally desirable to select a codon thatis rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNAsynthetase is expressed. For example, exemplary codons include nonsensecodon such as stop codons (amber, ochre, and opal), four or more basecodons and other natural three-base codons that are rarely or unused.

Specific selector codon(s) can be introduced into appropriate positionsin the ABP polynucleotide coding sequence using mutagenesis methodsknown in the art (including but not limited to, site-specificmutagenesis, cassette mutagenesis, restriction selection mutagenesis,etc.).

Methods for generating components of the protein biosynthetic machinery,such as O—RSs, O-tRNAs, and orthogonal O-tRNA/O—RS pairs that can beused to incorporate a non-naturally encoded amino acid are described inWang, L., et al., Science 292: 498-500 (2001); Chin, J. W., et al., J.Am. Chem. Soc. 124:9026-9027 (2002); Zhang, Z. et al., Biochemistry 42:6735-6746 (2003). Methods and compositions for the in vivo incorporationof non-naturally encoded amino acids are described in U.S. PatentApplication Publication 2003/0082575 (Ser. No. 10/126,927) which isincorporated by reference herein. Methods for selecting an orthogonaltRNA-tRNA synthetase pair for use in in vivo translation system of anorganism are also described in U.S. Patent Application Publications2003/0082575 (Ser. No. 10/126,927) and 2003/0108885 (Ser. No.10/126,931) which are incorporated by reference herein.

Methods for producing at least one recombinant orthogonal aminoacyl-tRNAsynthetase (O—RS) comprise: (a) generating a library of (optionallymutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS)from a first organism, including but not limited to, a prokaryoticorganism, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P.furiosus, P. horikoshii, A. pernix, T. thermophilus, or the like, or aeukaryotic organism; (b) selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that aminoacylate an orthogonal tRNA(O-tRNA) in the presence of a non-naturally encoded amino acid and anatural amino acid, thereby providing a pool of active (optionallymutant) RSs; and/or, (c) selecting (optionally through negativeselection) the pool for active RSs (including but not limited to, mutantRSs) that preferentially aminoacylate the O-tRNA in the absence of thenon-naturally encoded amino acid, thereby providing the at least onerecombinant O—RS; wherein the at least one recombinant O—RSpreferentially aminoacylates the O-tRNA with the non-naturally encodedamino acid.

In one embodiment, the RS is an inactive RS. The inactive RS can begenerated by mutating an active RS. For example, the inactive RS can begenerated by mutating at least about 1, at least about 2, at least about3, at least about 4, at least about 5, at least about 6, or at leastabout 10 or more amino acids to different amino acids, including but notlimited to, alanine.

Libraries of mutant RSs can be generated using various techniques knownin the art, including but not limited to rational design based onprotein three dimensional RS structure, or mutagenesis of RS nucleotidesin a random or rational design technique. For example, the mutant RSscan be generated by site-specific mutations, random mutations, diversitygenerating recombination mutations, chimeric constructs, rational designand by other methods described herein or known in the art.

In one embodiment, selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that are active, including but notlimited to, that aminoacylate an orthogonal tRNA (O-tRNA) in thepresence of a non-naturally encoded amino acid and a natural amino acid,includes: introducing a positive selection or screening marker,including but not limited to, an antibiotic resistance gene, or thelike, and the library of (optionally mutant) RSs into a plurality ofcells, wherein the positive selection and/or screening marker comprisesat least one selector codon, including but not limited to, an amber,ochre, or opal codon; growing the plurality of cells in the presence ofa selection agent; identifying cells that survive (or show a specificresponse) in the presence of the selection and/or screening agent bysuppressing the at least one selector codon in the positive selection orscreening marker, thereby providing a subset of positively selectedcells that contains the pool of active (optionally mutant) RSs.Optionally, the selection and/or screening agent concentration can bevaried.

In one aspect, the positive selection marker is a chloramphenicolacetyltransferase (CAT) gene and the selector codon is an amber stopcodon in the CAT gene. Optionally, the positive selection marker is aβ-lactamase gene and the selector codon is an amber stop codon in theβ-lactamase gene. In another aspect the positive screening markercomprises a fluorescent or luminescent screening marker or an affinitybased screening marker (including but not limited to, a cell surfacemarker).

In one embodiment, negatively selecting or screening the pool for activeRSs (optionally mutants) that preferentially aminoacylate the O-tRNA inthe absence of the non-naturally encoded amino acid includes:introducing a negative selection or screening marker with the pool ofactive (optionally mutant) RSs from the positive selection or screeninginto a plurality of cells of a second organism, wherein the negativeselection or screening marker comprises at least one selector codon(including but not limited to, an antibiotic resistance gene, includingbut not limited to, a chloramphenicol acetyltransferase (CAT) gene);and, identifying cells that survive or show a specific screeningresponse in a first medium supplemented with the non-naturally encodedamino acid and a screening or selection agent, but fail to survive or toshow the specific response in a second medium not supplemented with thenon-naturally encoded amino acid and the selection or screening agent,thereby providing surviving cells or screened cells with the at leastone recombinant O—RS. For example, a CAT identification protocoloptionally acts as a positive selection and/or a negative screening indetermination of appropriate O—RS recombinants. For instance, a pool ofclones is optionally replicated on growth plates containing CAT (whichcomprises at least one selector codon) either with or without one ormore non-naturally encoded amino acid. Colonies growing exclusively onthe plates containing non-naturally encoded amino acids are thusregarded as containing recombinant O—RS. In one aspect, theconcentration of the selection (and/or screening) agent is varied. Insome aspects the first and second organisms are different. Thus, thefirst and/or second organism optionally comprises: a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacterium, a eubacterium, a plant, an insect, a protist, etc. Inother embodiments, the screening marker comprises a fluorescent orluminescent screening marker or an affinity based screening marker.

In another embodiment, screening or selecting (including but not limitedto, negatively selecting) the pool for active (optionally mutant) RSsincludes: isolating the pool of active mutant RSs from the positiveselection step (b); introducing a negative selection or screeningmarker, wherein the negative selection or screening marker comprises atleast one selector codon (including but not limited to, a toxic markergene, including but not limited to, a ribonuclease barnase gene,comprising at least one selector codon), and the pool of active(optionally mutant) RSs into a plurality of cells of a second organism;and identifying cells that survive or show a specific screening responsein a first medium not supplemented with the non-naturally encoded aminoacid, but fail to survive or show a specific screening response in asecond medium supplemented with the non-naturally encoded amino acid,thereby providing surviving or screened cells with the at least onerecombinant O—RS, wherein the at least one recombinant O—RS is specificfor the non-naturally encoded amino acid. In one aspect, the at leastone selector codon comprises about two or more selector codons. Suchembodiments optionally can include wherein the at least one selectorcodon comprises two or more selector codons, and wherein the first andsecond organism are different (including but not limited to, eachorganism is optionally, including but not limited to, a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacteria, a eubacteria, a plant, an insect, a protist, etc.).Also, some aspects include wherein the negative selection markercomprises a ribonuclease barnase gene (which comprises at least oneselector codon). Other aspects include wherein the screening markeroptionally comprises a fluorescent or luminescent screening marker or anaffinity based screening marker. In the embodiments herein, thescreenings and/or selections optionally include variation of thescreening and/or selection stringency.

In one embodiment, the methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O—RS) can further comprise: (d)isolating the at least one recombinant O—RS; (e) generating a second setof O—RS (optionally mutated) derived from the at least one recombinantO—RS; and, (f) repeating steps (b) and (c) until a mutated O—RS isobtained that comprises an ability to preferentially aminoacylate theO-tRNA. Optionally, steps (d)-(f) are repeated, including but notlimited to, at least about two times. In one aspect, the second set ofmutated O—RS derived from at least one recombinant O—RS can be generatedby mutagenesis, including but not limited to, random mutagenesis,site-specific mutagenesis, recombination or a combination thereof.

The stringency of the selection/screening steps, including but notlimited to, the positive selection/screening step (b), the negativeselection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c), in the above-described methods,optionally includes varying the selection/screening stringency. Inanother embodiment, the positive selection/screening step (b), thenegative selection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c) comprise using a reporter, whereinthe reporter is detected by fluorescence-activated cell sorting (FACS)or wherein the reporter is detected by luminescence. Optionally, thereporter is displayed on a cell surface, on a phage display or the likeand selected based upon affinity or catalytic activity involving thenon-naturally encoded amino acid or an analogue. In one embodiment, themutated synthetase is displayed on a cell surface, on a phage display orthe like.

Methods for producing a recombinant orthogonal tRNA (O-tRNA) include:(a) generating a library of mutant tRNAs derived from at least one tRNA,including but not limited to, a suppressor tRNA, from a first organism;(b) selecting (including but not limited to, negatively selecting) orscreening the library for (optionally mutant) tRNAs that areaminoacylated by an aminoacyl-tRNA synthetase (RS) from a secondorganism in the absence of a RS from the first organism, therebyproviding a pool of tRNAs (optionally mutant); and, (c) selecting orscreening the pool of tRNAs (optionally mutant) for members that areaminoacylated by an introduced orthogonal RS (O—RS), thereby providingat least one recombinant O-tRNA; wherein the at least one recombinantO-tRNA recognizes a selector codon and is not efficiency recognized bythe RS from the second organism and is preferentially aminoacylated bythe O—RS. In some embodiments the at least one tRNA is a suppressor tRNAand/or comprises a unique three base codon of natural and/or unnaturalbases, or is a nonsense codon, a rare codon, an unnatural codon, a codoncomprising at least 4 bases, an amber codon, an ochre codon, or an opalstop codon. In one embodiment, the recombinant O-tRNA possesses animprovement of orthogonality. It will be appreciated that in someembodiments, O-tRNA is optionally imported into a first organism from asecond organism without the need for modification. In variousembodiments, the first and second organisms are either the same ordifferent and are optionally chosen from, including but not limited to,prokaryotes (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Escherichia coli, Halobacterium,etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria,plants, insects, protists, etc. Additionally, the recombinant tRNA isoptionally aminoacylated by a non-naturally encoded amino acid, whereinthe non-naturally encoded amino acid is biosynthesized in vivo eithernaturally or through genetic manipulation. The non-naturally encodedamino acid is optionally added to a growth medium for at least the firstor second organism.

In one aspect, selecting (including but not limited to, negativelyselecting) or screening the library for (optionally mutant) tRNAs thatare aminoacylated by an aminoacyl-tRNA synthetase (step (b)) includes:introducing a toxic marker gene, wherein the toxic marker gene comprisesat least one of the selector codons (or a gene that leads to theproduction of a toxic or static agent or a gene essential to theorganism wherein such marker gene comprises at least one selector codon)and the library of (optionally mutant) tRNAs into a plurality of cellsfrom the second organism; and, selecting surviving cells, wherein thesurviving cells contain the pool of (optionally mutant) tRNAs comprisingat least one orthogonal tRNA or nonfunctional tRNA. For example,surviving cells can be selected by using a comparison ratio cell densityassay.

In another aspect, the toxic marker gene can include two or moreselector codons. In another embodiment of the methods, the toxic markergene is a ribonuclease barnase gene, where the ribonuclease barnase genecomprises at least one amber codon. Optionally, the ribonuclease barnasegene can include two or more amber codons.

In one embodiment, selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O—RS) can include: introducing a positive selection orscreening marker gene, wherein the positive marker gene comprises a drugresistance gene (including but not limited to, β-lactamase gene,comprising at least one of the selector codons, such as at least oneamber stop codon) or a gene essential to the organism, or a gene thatleads to detoxification of a toxic agent, along with the O—RS, and thepool of (optionally mutant) tRNAs into a plurality of cells from thesecond organism; and, identifying surviving or screened cells grown inthe presence of a selection or screening agent, including but notlimited to, an antibiotic, thereby providing a pool of cells possessingthe at least one recombinant tRNA, where the at least one recombinanttRNA is aminoacylated by the O—RS and inserts an amino acid into atranslation product encoded by the positive marker gene, in response tothe at least one selector codons. In another embodiment, theconcentration of the selection and/or screening agent is varied.

Methods for generating specific O-tRNA/O—RS pairs are provided. Methodsinclude: (a) generating a library of mutant tRNAs derived from at leastone tRNA from a first organism; (b) negatively selecting or screeningthe library for (optionally mutant) tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (RS) from a second organism in the absence ofa RS from the first organism, thereby providing a pool of (optionallymutant) tRNAs; (c) selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O—RS), thereby providing at least one recombinant O-tRNA.The at least one recombinant O-tRNA recognizes a selector codon and isnot efficiency recognized by the RS from the second organism and ispreferentially aminoacylated by the O—RS. The method also includes (d)generating a library of (optionally mutant) RSs derived from at leastone aminoacyl-tRNA synthetase (RS) from a third organism; (e) selectingor screening the library of mutant RSs for members that preferentiallyaminoacylate the at least one recombinant O-tRNA in the presence of anon-naturally encoded amino acid and a natural amino acid, therebyproviding a pool of active (optionally mutant) RSs; and, (f) negativelyselecting or screening the pool for active (optionally mutant) RSs thatpreferentially aminoacylate the at least one recombinant O-tRNA in theabsence of the non-naturally encoded amino acid, thereby providing theat least one specific O-tRNA/O—RS pair, wherein the at least onespecific O-tRNA/O—RS pair comprises at least one recombinant O—RS thatis specific for the non-naturally encoded amino acid and the at leastone recombinant O-tRNA. Specific O-tRNA/O—RS pairs produced by themethods are included. For example, the specific O-tRNA/O—RS pair caninclude, including but not limited to, a mutRNATyr-mutTyrRS pair, suchas a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, amutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.Additionally, such methods include wherein the first and third organismare the same (including but not limited to, Methanococcus jannaschii).

Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use inan in vivo translation system of a second organism are also included inthe present invention. The methods include: introducing a marker gene, atRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from afirst organism into a first set of cells from the second organism;introducing the marker gene and the tRNA into a duplicate cell set froma second organism; and, selecting for surviving cells in the first setthat fail to survive in the duplicate cell set or screening for cellsshowing a specific screening response that fail to give such response inthe duplicate cell set, wherein the first set and the duplicate cell setare grown in the presence of a selection or screening agent, wherein thesurviving or screened cells comprise the orthogonal tRNA-tRNA synthetasepair for use in the in the in vivo translation system of the secondorganism. In one embodiment, comparing and selecting or screeningincludes an in vivo complementation assay. The concentration of theselection or screening agent can be varied.

The organisms of the present invention comprise a variety of organismand a variety of combinations. For example, the first and the secondorganisms of the methods of the present invention can be the same ordifferent. In one embodiment, the organisms are optionally a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, orthe like. Alternatively, the organisms optionally comprise a eukaryoticorganism, including but not limited to, plants (including but notlimited to, complex plants such as monocots, or dicots), algae,protists, fungi (including but not limited to, yeast, etc), animals(including but not limited to, mammals, insects, arthropods, etc.), orthe like. In another embodiment, the second organism is a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, T.thermophilus, or the like. Alternatively, the second organism can be aeukaryotic organism, including but not limited to, a yeast, a animalcell, a plant cell, a fungus, a mammalian cell, or the like. In variousembodiments the first and second organisms are different.

VI. Location of Non-Naturally-Occurring Amino Acids in ABP

The present invention contemplates incorporation of one or morenon-naturally-occurring amino acids into ABP. One or morenon-naturally-occurring amino acids may be incorporated at a particularposition which does not disrupt activity of the polypeptide. This can beachieved by making “conservative” substitutions, including but notlimited to, substituting hydrophobic amino acids with hydrophobic aminoacids, bulky amino acids for bulky amino acids, hydrophilic amino acidsfor hydrophilic amino acids) and/or inserting thenon-naturally-occurring amino acid in a location that is not requiredfor activity.

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-naturally encodedamino acid within the antigen-binding polypeptide. It is readilyapparent to those of ordinary skill in the art that any position of thepolypeptide chain is suitable for selection to incorporate anon-naturally encoded amino acid, and selection may be based on rationaldesign or by random selection for any or no particular desired purpose.Selection of desired sites may be for producing an ABP molecule havingany desired property or activity, including but not limited to,agonists, super-agonists, inverse agonists, antagonists, receptorbinding modulators, receptor activity modulators, dimer or multimerformation, no change to activity or property compared to the nativemolecule, or manipulating any physical or chemical property of thepolypeptide such as solubility, aggregation, or stability. For example,locations in the polypeptide required for biological activity of ABP canbe identified using alanine scanning or homolog scanning methods knownin the art. Residues other than those identified as critical tobiological activity by alanine or homolog scanning mutagenesis may begood candidates for substitution with a non-naturally encoded amino aciddepending on the desired activity sought for the polypeptide.Alternatively, the sites identified as critical to biological activitymay also be good candidates for substitution with a non-naturallyencoded amino acid, again depending on the desired activity sought forthe polypeptide. Another alternative would be to simply make serialsubstitutions in each position on the polypeptide chain with anon-naturally encoded amino acid and observe the effect on theactivities of the polypeptide. It is readily apparent to those ofordinary skill in the art that any means, technique, or method forselecting a position for substitution with a non-natural amino acid intoany polypeptide is suitable for use in the present invention.

Once residues that are likely to be intolerant to substitution withnon-naturally encoded amino acids have been eliminated, the impact ofproposed substitutions at each of the remaining positions can beexamined from the secondary, tertiary or quaternary structure, or thethree-dimensional crystal structure of the antigen-binding polypeptideand its binding partners. Thus, those of skill in the art can readilyidentify amino acid positions that can be substituted with non-naturallyencoded amino acids.

Exemplary residues of incorporation of a non-naturally encoded aminoacid include, but are not limited to, those that are excluded frompotential antigen binding regions, may be fully or partially solventexposed, have minimal or no hydrogen-bonding interactions with nearbyresidues, may be minimally exposed to nearby reactive residues, may beon one or more of the exposed faces of ABP, may be a site or sites ofABP that are juxtaposed to a second ABP, or other molecule or fragmentthereof, may be in regions that are highly flexible, or structurallyrigid, as predicted by the three-dimensional, secondary, tertiary, orquaternary structure of ABP, bound or unbound to its antigen, or coupledor not coupled to another ABP or other biologically active molecule, ormay modulate the conformation of the ABP itself or a dimer or multimercomprising one or more ABP, by altering the flexibility or rigidity ofthe complete structure as desired. Residues for incorporation ofnon-natural amino acids may be a part of a cleavage sequence, linkersequence joining antibody fragments or ABPs, antibody-binding domain(including but not limited to, myc tag, FLAG or poly-His) or otheraffinity based sequence (including but not limited to, FLAG, poly-His,GST, etc.). Residues for incorporation of a non-natural amino acid maybe N-terminal or C-terminal residues of an ABP or non-antigen bindingresidues of an ABP.

A wide variety of non-naturally encoded amino acids can be substitutedfor, or incorporated into, a given position in ABP. In general, aparticular non-naturally encoded amino acid is selected forincorporation based on an examination of the three dimensional crystalstructure of ABP with its antigen or the secondary, tertiary, orquaternary structure of ABP determined by any other means, a preferencefor conservative substitutions (i.e., aryl-based non-naturally encodedamino acids, such as p-acetylphenylalanine or O-propargyltyrosinesubstituting for Phe, Tyr or Trp), and the specific conjugationchemistry that one desires to introduce into the antigen-bindingpolypeptide (e.g., the introduction of 4-azidophenylalanine if one wantsto effect a Huisgen [3+2]cycloaddition with a water soluble polymerbearing an alkyne moiety or a amide bond formation with a water solublepolymer that bears an aryl ester that, in turn, incorporates a phosphinemoiety).

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a water-soluble dendrimer, a cyclodextrin, an inhibitoryribonucleic acid, a biomaterial, a nanoparticle, a spin label, afluorophore, a metal-containing moiety, a radioactive moiety, a novelfunctional group, a group that covalently or noncovalently interactswith other molecules, a photocaged moiety, a photoisomerizable moiety,biotin, a derivative of biotin, a derivative of biotin, a biotinanalogue, a moiety incorporating a heavy atom, a chemically cleavablegroup, a photocleavable group, an elongated side chain, a carbon-linkedsugar, a redox-active agent, an amino thioacid, a toxic moiety, anisotopically labeled moiety, a biophysical probe, a phosphorescentgroup, a chemiluminescent group, an electron dense group, a magneticgroup, an intercalating group, a chromophore, an energy transfer agent,a biologically active agent, a detectable label, a small molecule, orany combination of the above, or any other desirable compound orsubstance) that comprises a second reactive group. The first reactivegroup reacts with the second reactive group to attach the molecule tothe unnatural amino acid through a [3+2]cycloaddition. In oneembodiment, the first reactive group is an alkynyl or azido moiety andthe second reactive group is an azido or alkynyl moiety. For example,the first reactive group is the alkynyl moiety (including but notlimited to, in unnatural amino acid p-propargyloxyphenylalanine) and thesecond reactive group is the azido moiety. In another example, the firstreactive group is the azido moiety (including but not limited to, in theunnatural amino acid p-azido-L-phenylalanine) and the second reactivegroup is the alkynyl moiety.

In some cases, the non-naturally encoded amino acid substitution(s) willbe combined with other additions, substitutions or deletions within theantigen-binding polypeptide to affect other biological traits of ABP. Insome cases, the other additions, substitutions or deletions may increasethe stability (including but not limited to, resistance to proteolyticdegradation) of the ABP or increase affinity of the ABP for an ABPreceptor or antigen. In some cases, the other additions, substitutionsor deletions may increase the solubility (including but not limited to,when expressed in E. coli or other host cells) of the antigen-bindingpolypeptide. In some embodiments additions, substitutions or deletionsmay increase the polypeptide solubility following expression in E. colior other recombinant host cells. In some embodiments sites are selectedfor substitution with a naturally encoded or non-natural amino acid inaddition to another site for incorporation of a non-natural amino acidthat results in increasing the polypeptide solubility followingexpression in E. coli or other recombinant host cells. In someembodiments, the antigen-binding polypeptides comprise another addition,substitution or deletion that modulates affinity for the ABP receptor,modulates (including but not limited to, increases or decreases)receptor dimerization, stabilizes receptor dimers, modulates circulatinghalf-life, modulates release or bio-availability, facilitatespurification, or improves or alters a particular route ofadministration. Similarly, antigen-binding polypeptides can comprisechemical or enzyme cleavage sequences, protease cleavage sequences,reactive groups, antibody-binding domains (including but not limited to,FLAG or poly-His) or other affinity based sequences (including, but notlimited to, FLAG, poly-His, GST, etc.) or linked molecules (including,but not limited to, biotin) that improve detection (including, but notlimited to, GFP), purification, transport through tissues or cellmembranes, prodrug release or activation, ABP size reduction, or othertraits of the polypeptide.

In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids aresubstituted with one or more non-naturally-encoded amino acids. In somecases, the ABP further includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moresubstitutions of one or more non-naturally encoded amino acids fornaturally-occurring amino acids. In some embodiments, at least tworesidues in the following regions of ABP are substituted with one ormore non-naturally encoded amino acids. In some cases, the two or morenon-naturally encoded residues are linked to one or more lower molecularweight linear or branched PEGs (approximately ˜5-20 kDa in mass orless), thereby enhancing binding affinity and comparable serum half-liferelative to the species attached to a single, higher molecular weightPEG.

In some embodiments, up to two of the residues of an antigen-bindingpolypeptide are substituted with one or more non-naturally-encoded aminoacids.

VII. Expression in Non-Eukaryotes and Eukaryotes

To obtain high level expression of a cloned ABP polynucleotide, onetypically subclones polynucleotides encoding an antigen-bindingpolypeptide of the invention into an expression vector that contains astrong promoter to direct transcription, a transcription/translationterminator, and if for a nucleic acid encoding a protein, a ribosomebinding site for translational initiation. Suitable bacterial promotersare well known in the art and described, e.g., in Sambrook et al. andAusubel et al.

Bacterial expression systems for expressing ABP polypeptides of theinvention are available in, including but not limited to, E. coli,Bacillus sp., Pseudomonas fluorescens, Pseudomonas aeruginosa,Pseudomonas putida, and Salmonella (Palva et al., Gene 22:229-235(1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are well known inthe art and are also commercially available. In cases where orthogonaltRNAs and aminoacyl tRNA synthetases (described above) are used toexpress the antigen-binding polypeptides of the invention, host cellsfor expression are selected based on their ability to use the orthogonalcomponents. Exemplary host cells include Gram-positive bacteria(including but not limited to B. brevis, B. subtilis, or Streptomyces)and Gram-negative bacteria (E. coli, Pseudomonas fluorescens,Pseudomonas aeruginosa, Pseudomonas putida), as well as yeast and othereukaryotic cells. Cells comprising O-tRNA/O—RS pairs can be used asdescribed herein.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to synthesize proteins that compriseunnatural amino acids in large useful quantities. In one aspect, thecomposition optionally includes, including but not limited to, at least10 micrograms, at least 50 micrograms, at least 75 micrograms, at least100 micrograms, at least 200 micrograms, at least 250 micrograms, atleast 500 micrograms, at least 1 milligram, at least 10 milligrams, atleast 100 milligrams, at least one gram, or more of the protein thatcomprises an unnatural amino acid, or an amount that can be achievedwith in vivo protein production methods (details on recombinant proteinproduction and purification are provided herein). In another aspect, theprotein is optionally present in the composition at a concentration of,including but not limited to, at least 10 micrograms of protein perliter, at least 50 micrograms of protein per liter, at least 75micrograms of protein per liter, at least 100 micrograms of protein perliter, at least 200 micrograms of protein per liter, at least 250micrograms of protein per liter, at least 500 micrograms of protein perliter, at least 1 milligram of protein per liter, or at least 10milligrams of protein per liter or more, in, including but not limitedto, a cell lysate, a buffer, a pharmaceutical buffer, or other liquidsuspension (including but not limited to, in a volume of, including butnot limited to, anywhere from about 1 nl to about 100 L). The productionof large quantities (including but not limited to, greater that thattypically possible with other methods, including but not limited to, invitro translation) of a protein in a eukaryotic cell including at leastone unnatural amino acid is a feature of the invention.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to biosynthesize proteins that compriseunnatural amino acids in large useful quantities. For example, proteinscomprising an unnatural amino acid can be produced at a concentrationof, including but not limited to, at least 10 μg/liter, at least 50μg/liter, at least 75 μg/liter, at least 100 μg/liter, at least 200μg/liter, at least 250 μg/liter, or at least 500 μg/liter, at least 1mg/liter, at least 2 mg/liter, at least 3 mg/liter, at least 4 mg/liter,at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more of protein in a cellextract, cell lysate, culture medium, a buffer, and/or the like.

I. Expression Systems, Culture and Isolation

ABP may be expressed in any number of suitable expression systemsincluding, for example, yeast, insect cells, mammalian cells, andbacteria. A description of exemplary expression systems is providedbelow.

Yeast As used herein, the term “yeast” includes any of the variousyeasts capable of expressing a gene encoding ABP. Such yeasts include,but are not limited to, ascosporogenous yeasts (Endomycetales),basidiosporogenous yeasts and yeasts belonging to the Fungi imperfecti(Blastomycetes) group. The ascosporogenous yeasts are divided into twofamilies, Spernophthoraceae and Saccharomycetaceae. The latter iscomprised of four subfamilies, Schizosaccharomycoideae (e.g., genusSchizosaccharomyces), Nadsonioideae, Lipomycoideae and Saccharomycoideae(e.g., genera Pichia, Kluyveromyces and Saccharomyces). Thebasidiosporogenous yeasts include the genera Leucosporidium,Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeastsbelonging to the Fungi Imperfecti (Blastomycetes) group are divided intotwo families, Sporobolomycetaceae (e.g., genera Sporobolomyces andBullera) and Cryptococcaceae (e.g., genus Candida).

Of particular interest for use with the present invention are specieswithin the genera Pichia, Kluyveromyces, Saccharomyces,Schizosaccharomyces, Hansenula, Torulopsis, and Candida, including, butnot limited to, P. pastoris, P. guillerimondii, S. cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S. norbensis,S. oviformis, K. lactis, K. fragilis, C. albicans, C. maltosa, and H.polymorpha.

The selection of suitable yeast for expression of ABP is within theskill of one of ordinary skill in the art. In selecting yeast hosts forexpression, suitable hosts may include those shown to have, for example,good secretion capacity, low proteolytic activity, good secretioncapacity, good soluble protein production, and overall robustness. Yeastare generally available from a variety of sources including, but notlimited to, the Yeast Genetic Stock Center, Department of Biophysics andMedical Physics, University of California (Berkeley, Calif.), and theAmerican Type Culture Collection (“ATCC”) (Manassas, Va.).

The term “yeast host” or “yeast host cell” includes yeast that can be,or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original yeast hostcell that has received the recombinant vectors or other transfer DNA. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding ABP, are included in theprogeny intended by this definition.

Expression and transformation vectors, including extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeast hosts. For example, expression vectors have beendeveloped for S. cerevisiae (Sikorski et al., GENETICS (1998) 112:19;Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC. NATL.ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL.BIOL. (1986) 6:142); C. maltosa (Kunze et al., J. BASIC MICROBIOL.(1985) 25:141); H. polymorpha (Gleeson et al., J. GEN. MICROBIOL. (1986)132:3459; Roggenkamp et al., MOL. GEN. GENET. (1986) 202:302); K.fragilis (Das et al., J. BACTERIOL. (1984) 158:1165); K. lactis (DeLouvencourt et al., J. BACTERIOL. (1983) 154:737; Van den Berg et al.,BIO/TECHNOLOGY (1990) 8:135); P. guillerimondii (Kunze et al., J. BASICMICROBIOL. (1985) 25:141); P. pastoris (U.S. Pat. Nos. 5,324,639;4,929,555; and 4,837,148; Cregg et al., MOL. CELL. BIOL. (1985) 5:3376);Schizosaccharomyces pombe (Beach and Nurse, NATURE (1981) 300:706); andY. lipolytica (Davidow et al., CURR. GENET. (1985) 10:380 (1985);Gaillardin et al., CURR. GENET. (1985) 10:49); A. nidulans (Ballance etal., BIOCHEM. BIOPHYS. RES. COMMUN. (1983) 112:284-89; Tilburn et al.,GENE (1983) 26:205-221; and Yelton et al., PROC. NATL. ACAD. SCI. USA(1984) 81:1470-74); A. niger (Kelly and Hynes, EMBO J. (1985)4:475-479); T. reesia (EP 0 244 234); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357), eachincorporated by reference herein.

Control sequences for yeast vectors are well known to those of ordinaryskill in the art and include, but are not limited to, promoter regionsfrom genes such as alcohol dehydrogenase (ADH) (EP 0 284 044); enolase;glucokinase; glucose-6-phosphate isomerase;glyceraldehydes-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase;phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase(PyK) (EP 0 329 203). The yeast PHO5 gene, encoding acid phosphatase,also may provide useful promoter sequences (Myanohara et al., PROC.NATL. ACAD. SCI. USA (1983) 80:1). Other suitable promoter sequences foruse with yeast hosts may include the promoters for 3-phosphoglyceratekinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255:2073); and otherglycolytic enzymes, such as pyruvate decarboxylase, triosephosphateisomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY(1978) 17:4900; Hess et al., J. ADV. ENZYME REG. (1968) 7:149).Inducible yeast promoters having the additional advantage oftranscription controlled by growth conditions may include the promoterregions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase;metallothionein; glyceraldehyde-3-phosphate dehydrogenase; degradativeenzymes associated with nitrogen metabolism; and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 0 073 657.

Yeast enhancers also may be used with yeast promoters. In addition,synthetic promoters may also function as yeast promoters. For example,the upstream activating sequences (UAS) of a yeast promoter may bejoined with the transcription activation region of another yeastpromoter, creating a synthetic hybrid promoter. Examples of such hybridpromoters include the ADH regulatory sequence linked to the GAPtranscription activation region. See U.S. Pat. Nos. 4,880,734 and4,876,197, which are incorporated by reference herein. Other examples ofhybrid promoters include promoters that consist of the regulatorysequences of the ADH2, GAL4, GAL10, or PHO5 genes, combined with thetranscriptional activation region of a glycolytic enzyme gene such asGAP or PyK. See EP 0 164 556. Furthermore, a yeast promoter may includenaturally occurring promoters of non-yeast origin that have the abilityto bind yeast RNA polymerase and initiate transcription.

Other control elements that may comprise part of the yeast expressionvectors include terminators, for example, from GAPDH or the enolasegenes (Holland et al., J. BIOL. CHEM. (1981) 256:1385). In addition, theorigin of replication from the 2μ plasmid origin is suitable for yeast.A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid. See Tschemper et al., GENE (1980) 10:157; Kingsman etal., GENE (1979) 7:141. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

Methods of introducing exogenous DNA into yeast hosts are well known tothose of ordinary skill in the art, and typically include, but are notlimited to, either the transformation of spheroplasts or of intact yeasthost cells treated with alkali cations. For example, transformation ofyeast can be carried out according to the method described in Hsiao etal., PROC. NATL. ACAD. SCI. USA (1979) 76:3829 and Van Solingen et al.,J. BACT. (1977) 130:946. However, other methods for introducing DNA intocells such as by nuclear injection, electroporation, or protoplastfusion may also be used as described generally in SAMBROOK ET AL.,MOLECULAR CLONING: A LAB. MANUAL (2001). Yeast host cells may then becultured using standard techniques known to those of ordinary skill inthe art.

Other methods for expressing heterologous proteins in yeast host cellsare well known to those of ordinary skill in the art. See generally U.S.Patent Publication No. 20020055169, U.S. Pat. Nos. 6,361,969; 6,312,923;6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; and5,089,398; U.S. Reexamined Patent Nos. RE37,343 and RE35,749; PCTPublished Patent Applications WO 99/078621; WO 98/37208; and WO98/26080; European Patent Applications EP 0 946 736; EP 0 732 403; EP 0480 480; EP 0 460 071; EP 0 340 986; EP 0 329 203; EP 0 324 274; and EP0 164 556. See also Gellissen et al., ANTONME VAN LEEUWENHOEK (1992)62(1-2):79-93; Romanos et al., YEAST (1992) 8(6):423-488; Goeddel,METHODS IN ENZYMOLOGY (1990) 185:3-7, each incorporated by referenceherein.

The yeast host strains may be grown in fermentors during theamplification stage using standard feed batch fermentation methods wellknown to those of ordinary skill in the art. The fermentation methodsmay be adapted to account for differences in a particular yeast host'scarbon utilization pathway or mode of expression control. For example,fermentation of a Saccharomyces yeast host may require a single glucosefeed, complex nitrogen source (e.g., casein hydrolysates), and multiplevitamin supplementation. In contrast, the methylotrophic yeast P.pastoris may require glycerol, methanol, and trace mineral feeds, butonly simple ammonium (nitrogen) salts for optimal growth and expression.See, e.g., U.S. Pat. No. 5,324,639; Elliott et al., J. PROTEIN CHEM.(1990) 9:95; and Fieschko et al., BIOTECH. BIOENG. (1987) 29:1113,incorporated by reference herein.

Such fermentation methods, however, may have certain common featuresindependent of the yeast host strain employed. For example, a growthlimiting nutrient, typically carbon, may be added to the fermentorduring the amplification phase to allow maximal growth. In addition,fermentation methods generally employ a fermentation medium designed tocontain adequate amounts of carbon, nitrogen, basal salts, phosphorus,and other minor nutrients (vitamins, trace minerals and salts, etc.).Examples of fermentation media suitable for use with Pichia aredescribed in U.S. Pat. Nos. 5,324,639 and 5,231,178, which areincorporated by reference herein.

Baculovirus-Infected Insect Cells The term “insect host” or “insect hostcell” refers to a insect that can be, or has been, used as a recipientfor recombinant vectors or other transfer DNA. The term includes theprogeny of the original insect host cell that has been transfected. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding ABP, are included in theprogeny intended by this definition.

The selection of suitable insect cells for expression of ABP is wellknown to those of ordinary skill in the art. Several insect species arewell described in the art and are commercially available including Aedesaegypti, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda,and Trichoplusia ni. In selecting insect hosts for expression, suitablehosts may include those shown to have, inter alia, good secretioncapacity, low proteolytic activity, and overall robustness. Insect aregenerally available from a variety of sources including, but not limitedto, the Insect Genetic Stock Center, Department of Biophysics andMedical Physics, University of California (Berkeley, Calif.); and theAmerican Type Culture Collection (“ATCC”) (Manassas, Va.).

Generally, the components of a baculovirus-infected insect expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene to be expressed;a wild type baculovirus with sequences homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.The materials, methods and techniques used in constructing vectors,transfecting cells, picking plaques, growing cells in culture, and thelike are known in the art and manuals are available describing thesetechniques.

After inserting the heterologous gene into the transfer vector, thevector and the wild type viral genome are transfected into an insecthost cell where the vector and viral genome recombine. The packagedrecombinant virus is expressed and recombinant plaques are identifiedand purified. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, forexample, Invitrogen Corp. (Carlsbad, Calif.). These techniques aregenerally known to those skilled in the art and fully described inSUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN NO.1555 (1987), herein incorporated by reference. See also, RICHARDSON, 39METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSION PROTOCOLS (1995);AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 16.9-16.11(1994); KING AND POSSEE, THE BACULOVIRUS SYSTEM: A LABORATORY GUIDE(1992); and O'REILLY ET AL., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992).

Indeed, the production of various heterologous proteins usingbaculovirus/insect cell expression systems is well known in the art.See, e.g., U.S. Pat. Nos. 6,368,825; 6,342,216; 6,338,846; 6,261,805;6,245,528, 6,225,060; 6,183,987; 6,168,932; 6,126,944; 6,096,304;6,013,433; 5,965,393; 5,939,285; 5,891,676; 5,871,986; 5,861,279;5,858,368; 5,843,733; 5,762,939; 5,753,220; 5,605,827; 5,583,023;5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO 01/90390; WO 01/27301;WO 01/05956; WO 00/55345; WO 00/20032 WO 99/51721; WO 99/45130; WO99/31257; WO 99/10515; WO 99/09193; WO 97/26332; WO 96/29400; WO96/25496; WO 96/06161; WO 95/20672; WO 93/03173; WO 92/16619; WO92/03628; WO 92/01801; WO 90/14428; WO 90/10078; WO 90/02566; WO90/02186; WO 90/01556; WO 89/01038; WO 89/01037; WO 88/07082, which areincorporated by reference herein.

Vectors that are useful in baculovirus/insect cell expression systemsare known in the art and include, for example, insect expression andtransfer vectors derived from the baculovirus Autographa californicanuclear polyhedrosis virus (AcNPV), which is a helper-independent, viralexpression vector. Viral expression vectors derived from this systemusually use the strong viral polyhedrin gene promoter to driveexpression of heterologous genes. See generally, Reilly ET AL.,BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Prior to inserting the foreign gene into the baculovirus genome, theabove-described components, comprising a promoter, leader (if desired),coding sequence of interest, and transcription termination sequence, aretypically assembled into an intermediate transplacement construct(transfer vector). Intermediate transplacement constructs are oftenmaintained in a replicon, such as an extra chromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as bacteria. Thereplicon will have a replication system, thus allowing it to bemaintained in a suitable host for cloning and amplification. Morespecifically, the plasmid may contain the polyhedrin polyadenylationsignal (Miller et al., ANN. REV. MICROBIOL. (1988) 42:177) and aprokaryotic ampicillin-resistance (amp) gene and origin of replicationfor selection and propagation in E. coli.

One commonly used transfer vector for introducing foreign genes intoAcNPV is pAc373. Many other vectors, known to those of skill in the art,have also been designed including, for example, pVL985, which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 base pairs downstream from the ATT. See Luckow andSummers, 17 VIROLOGY 31 (1989). Other commercially available vectorsinclude, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac;pBlueBac4.5 (Invitrogen Corp., Carlsbad, Calif.).

After insertion of the heterologous gene, the transfer vector and wildtype baculoviral genome are co-transfected into an insect cell host.Methods for introducing heterologous DNA into the desired site in thebaculovirus virus are known in the art. See SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987); Smith et al.,MOL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989)17:31. For example, the insertion can be into a gene such as thepolyhedrin gene, by homologous double crossover recombination; insertioncan also be into a restriction enzyme site engineered into the desiredbaculovirus gene. See Miller et al., BIOESSAYS (1989) 4:91.

Transfection may be accomplished by electroporation. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Mann and King, J. GEN.VIROL. (1989) 70:3501. Alternatively, liposomes may be used to transfectthe insect cells with the recombinant expression vector and thebaculovirus. See, e.g., Liebman et al., BIOTECHNIQUES (1999) 26(1):36;Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL.CHEM. (1998) 273(22):13570; Schmidt et al., PROTEIN EXPRESSION ANDPURIFICATION (1998) 12:323; Siffert et al., NATURE GENETICS (1998)18:45; TILKINS ET AL., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154(1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263;Dolphin et al., NATURE GENETICS (1997) 17:491; Kost et al., GENE (1997)190:139; Jakobsson et al., J. BIOL. CHEM. (1996) 271:22203; Rowles etal., J. BIOL. CHEM. (1996) 271(37):22376; Reversey et al., J. BIOL.CHEM. (1996) 271(39):23607-10; Stanley et al., J. BIOL. CHEM. (1995)270:4121; Sisk et al., J. VIROL. (1994) 68(2):766; and Peng et al.,BIOTECHNIQUES (1993) 14.2:274. Commercially available liposomes include,for example, Cellfectin® and Lipofectin® (Invitrogen, Corp., Carlsbad,Calif.). In addition, calcium phosphate transfection may be used. SeeTROTTER AND WOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR(1990) 18(19):5667; and Mann and King, J. GEN. VIROL. (1989) 70:3501.

Baculovirus expression vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g., structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus promoter may also have asecond domain called an enhancer, which, if present, is usually distalto the structural gene. Moreover, expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in the infectioncycle, provide particularly useful promoter sequences. Examples includesequences derived from the gene encoding the viral polyhedron protein(FRIESEN ET AL ., The Regulation of Baculovirus Gene Expression in THEMOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476)and the gene encoding the p10 protein (Vlak et al., J. GEN. VIROL.(1988) 69:765).

The newly formed baculovirus expression vector is packaged into aninfectious recombinant baculovirus and subsequently grown plaques may bepurified by techniques known to those skilled in the art. See Miller etal., BIOESSAYS (1989) 4:91; SUMMERS AND SMITH, TEXAS AGRICULTURALEXPERIMENT STATION BULLETIN NO. 1555 (1987).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia, Aedes aegypti (ATCCNo. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster(ATCC No. 1963), Spodoptera frugiperda, and Trichoplusia ni. See WO89/046,699; Wright, NATURE (1986) 321:718; Carbonell et al., J. VIROL.(1985) 56:153; Smith et al., MOL. CELL. BIOL. (1983) 3:2156. Seegenerally, Fraser et al., IN VITRO CELL. DEV. BIOL. (1989) 25:225. Morespecifically, the cell lines used for baculovirus expression vectorsystems commonly include, but are not limited to, Sf9 (Spodopterafrugiperda) (ATCC No. CRL-1711), Sf21 (Spodoptera frugiperda)(Invitrogen Corp., Cat. No. 11497-013 (Carlsbad, Calif.)), Tri-368(Trichoplusia ni), and High-Five™ BTI-TN-5B1-4 (Trichoplusia ni).

Cells and culture media are commercially available for both direct andfusion expression of heterologous polypeptides in abaculovirus/expression, and cell culture technology is generally knownto those skilled in the art.

E. Coli, Pseudomonas species, and other Prokaryotes Bacterial expressiontechniques are well known in the art. A wide variety of vectors areavailable for use in bacterial hosts. The vectors may be single copy orlow or high multicopy vectors. Vectors may serve for cloning and/orexpression. In view of the ample literature concerning vectors,commercial availability of many vectors, and even manuals describingvectors and their restriction maps and characteristics, no extensivediscussion is required here. As is well-known, the vectors normallyinvolve markers allowing for selection, which markers may provide forcytotoxic agent resistance, prototrophy or immunity. Frequently, aplurality of markers is present, which provide for differentcharacteristics.

A bacterial promoter is any DNA sequence capable of binding bacterialRNA polymerase and initiating the downstream (3′) transcription of acoding sequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site and a transcriptioninitiation site. A bacterial promoter may also have a second domaincalled an operator, that may overlap an adjacent RNA polymerase bindingsite at which RNA synthesis begins. The operator permits negativeregulated (inducible) transcription, as a gene repressor protein maybind the operator and thereby inhibit transcription of a specific gene.Constitutive expression may occur in the absence of negative regulatoryelements, such as the operator. In addition, positive regulation may beachieved by a gene activator protein binding sequence, which, if presentis usually proximal (5′) to the RNA polymerase binding sequence. Anexample of a gene activator protein is the catabolite activator protein(CAP), which helps initiate transcription of the lac operon inEscherichia coli (E. coli) [Raibaud et al., ANNU. REV. GENET. (1984)18:173]. Regulated expression may therefore be either positive ornegative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal., NATURE (1977) 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al., NUC. ACIDS RES. (1980) 8:4057; Yelverton et al.,NUCL. ACIDS RES. (1981) 9:731; U.S. Pat. No. 4,738,921; EP Pub. Nos. 036776 and 121 775, which are incorporated by reference herein]. Theβ-galactosidase (bla) promoter system [Weissmann (1981) “The cloning ofinterferon and other mistakes.” In Interferon 3 (Ed. I. Gresser)],bacteriophage lambda PL [Shimatake et al., NATURE (1981) 292:128] and T5[U.S. Pat. No. 4,689,406, which are incorporated by reference herein]promoter systems also provide useful promoter sequences. Preferredmethods of the present invention utilize strong promoters, such as theT7 promoter to induce ABP at high levels. Examples of such vectors arewell known in the art and include the pET29 series from Novagen, and thepPOP vectors described in WO99/05297, which is incorporated by referenceherein. Such expression systems produce high levels of ABP in the hostwithout compromising host cell viability or growth parameters. pET19(Novagen) is another vector known in the art.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433, which isincorporated by reference herein]. For example, the tac promoter is ahybrid trp-lac promoter comprised of both trp promoter and lac operonsequences that is regulated by the lac repressor [Amann et al., GENE(1983) 25:167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80:21].Furthermore, a bacterial promoter can include naturally occurringpromoters of non-bacterial origin that have the ability to bindbacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al., J.MOL. BIOL. (1986) 189:113; Tabor et al., Proc Natl Acad. Sci. (1985)82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EP Pub. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al., NATURE (1975) 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. “Genetic signals and nucleotide sequences inmessenger RNA”, In Biological Regulation and Development: GeneExpression (Ed. R. F. Goldberger, 1979)]. To express eukaryotic genesand prokaryotic genes with weak ribosome-binding site [Sambrook et al.“Expression of cloned genes in Escherichia coli”, Molecular Cloning: ALaboratory Manual, 1989].

The term “bacterial host” or “bacterial host cell” refers to a bacterialthat can be, or has been, used as a recipient for recombinant vectors orother transfer DNA. The term includes the progeny of the originalbacterial host cell that has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement to theoriginal parent, due to accidental or deliberate mutation. Progeny ofthe parental cell that are sufficiently similar to the parent to becharacterized by the relevant property, such as the presence of anucleotide sequence encoding ABP, are included in the progeny intendedby this definition.

The selection of suitable host bacteria for expression of ABP is wellknown to those of ordinary skill in the art. In selecting bacterialhosts for expression, suitable hosts may include those shown to have,inter alia, good inclusion body formation capacity, low proteolyticactivity, and overall robustness. Bacterial hosts are generallyavailable from a variety of sources including, but not limited to, theBacterial Genetic Stock Center, Department of Biophysics and MedicalPhysics, University of California (Berkeley, Calif.); and the AmericanType Culture Collection (“ATCC”) (Manassas, Va.).Industrial/pharmaceutical fermentation generally use bacterial derivedfrom K strains (e.g. W3110) or from bacteria derived from B strains(e.g. BL21). These strains are particularly useful because their growthparameters are extremely well known and robust. In addition, thesestrains are non-pathogenic, which is commercially important for safetyand environmental reasons. In one embodiment of the methods of thepresent invention, the E. coli host is a strain of BL21. In anotherembodiment of the methods of the present invention, the E. coli host isa protease minus strain including, but not limited to, OMP- and LON-. Inanother embodiment of the methods of the present invention, the hostcell strain is a species of Pseudomonas, including but not limited to,Pseudomonas fluorescens, Pseudomonas aeruginosa, and Pseudomonas putida.Pseudomonas fluorescens biovar 1, designated strain MB101, is known tobe useful for recombinant production and is available for therapeuticprotein production processes. An example of a Pseudomonas expressionsystem includes the system available from The Dow Chemical Company as ahost strain (Midland, Mich. available on the World Wide Web at dow.com).U.S. Pat. Nos. 4,755,465 and 4,859,600, which are incorporated byreference herein, describes the use of Pseudomonas strains as a hostcell for human growth hormone production.

Once a recombinant host cell strain has been established (i.e., theexpression construct has been introduced into the host cell and hostcells with the proper expression construct are isolated), therecombinant host cell strain is cultured under conditions appropriatefor production of ABP. As will be apparent to one of skill in the art,the method of culture of the recombinant host cell strain will bedependent on the nature of the expression construct utilized and theidentity of the host cell. Recombinant host strains are normallycultured using methods that are well known to the art. Recombinant hostcells are typically cultured in liquid medium containing assimilatablesources of carbon, nitrogen, and inorganic salts and, optionally,containing vitamins, amino acids, growth factors, and otherproteinaceous culture supplements well known to the art. Liquid mediafor culture of host cells may optionally contain antibiotics oranti-fungals to prevent the growth of undesirable microorganisms and/orcompounds including, but not limited to, antibiotics to select for hostcells containing the expression vector.

Recombinant host cells may be cultured in batch or continuous formats,with either cell harvesting (in the case where the ABP accumulatesintracellularly) or harvesting of culture supernatant in either batch orcontinuous formats. For production in prokaryotic host cells, batchculture and cell harvest are preferred.

The antigen-binding polypeptides of the present invention are normallypurified after expression in recombinant systems. The ABP may bepurified from host cells by a variety of methods known to the art.Normally, ABP produced in bacterial host cells is poorly soluble orinsoluble (in the form of inclusion bodies). In one embodiment of thepresent invention, amino acid substitutions may readily be made in theantigen-binding polypeptide that are selected for the purpose ofincreasing the solubility of the recombinantly produced proteinutilizing the methods disclosed herein as well as those known in theart. In the case of insoluble protein, the protein may be collected fromhost cell lysates by centrifugation and may further be followed byhomogenization of the cells. In the case of poorly soluble protein,compounds including, but not limited to, polyethylene imine (PEI) may beadded to induce the precipitation of partially soluble protein. Theprecipitated protein may then be conveniently collected bycentrifugation. Recombinant host cells may be disrupted or homogenizedto release the inclusion bodies from within the cells using a variety ofmethods well known to those of ordinary skill in the art. Host celldisruption or homogenization may be performed using well knowntechniques including, but not limited to, enzymatic cell disruption,sonication, dounce homogenization, or high pressure release disruption.In one embodiment of the method of the present invention, the highpressure release technique is used to disrupt the E. coli host cells torelease the inclusion bodies of ABP. When handling inclusion bodies ofABP, it is advantageous to minimize the homogenization time onrepetitions in order to maximize the yield of inclusion bodies withoutloss due to factors such as solubilization, mechanical shearing orproteolysis.

Insoluble or precipitated ABP may then be solubilized using any of anumber of suitable solubilization agents known to the art. Preferably,ABP is solubilized with urea or guanidine hydrochloride. The volume ofthe solubilized ABP should be minimized so that large batches may beproduced using conveniently manageable batch sizes. This factor may besignificant in a large-scale commercial setting where the recombinanthost may be grown in batches that are thousands of liters in volume. Inaddition, when manufacturing ABP in a large-scale commercial setting, inparticular for human pharmaceutical uses, the avoidance of harshchemicals that can damage the machinery and container, or the proteinproduct itself, should be avoided, if possible. It has been shown in themethod of the present invention that the milder denaturing agent ureacan be used to solubilize the ABP inclusion bodies in place of theharsher denaturing agent guanidine hydrochloride. The use of ureasignificantly reduces the risk of damage to stainless steel equipmentutilized in the manufacturing and purification process of ABP whileefficiently solubilizing the ABP inclusion bodies.

In the case of soluble ABP, ABP may be secreted into the periplasmicspace or into the culture medium. In addition, soluble ABP may bepresent in the cytoplasm of the host cells. It may be desired toconcentrate soluble ABP prior to performing purification steps. Standardtechniques known to those skilled in the art may be used to concentratesoluble ABP from, for example, cell lysates or culture medium. Inaddition, standard techniques known to those skilled in the art may beused to disrupt host cells and release soluble ABP from the cytoplasm orperiplasmic space of the host cells.

When ABP is produced as a fusion protein, the fusion sequence ispreferably removed. Removal of a fusion sequence may be accomplished byenzymatic or chemical cleavage, preferably by enzymatic cleavage.Enzymatic removal of fusion sequences may be accomplished using methodswell known to those in the art. The choice of enzyme for removal of thefusion sequence will be determined by the identity of the fusion, andthe reaction conditions will be specified by the choice of enzyme aswill be apparent to one skilled in the art. The cleaved ABP ispreferably purified from the cleaved fusion sequence by well knownmethods. Such methods will be determined by the identity and propertiesof the fusion sequence and the ABP, as will be apparent to one skilledin the art. Methods for purification may include, but are not limitedto, size-exclusion chromatography, hydrophobic interactionchromatography, ion-exchange chromatography or dialysis or anycombination thereof.

The ABP is also preferably purified to remove DNA from the proteinsolution. DNA may be removed by any suitable method known to the art,such as precipitation or ion exchange chromatography, but is preferablyremoved by precipitation with a nucleic acid precipitating agent, suchas, but not limited to, protamine sulfate. ABP may be separated from theprecipitated DNA using standard well known methods including, but notlimited to, centrifugation or filtration. Removal of host nucleic acidmolecules is an important factor in a setting where the ABP is to beused to treat humans and the methods of the present invention reducehost cell DNA to pharmaceutically acceptable levels.

Methods for small-scale or large-scale fermentation can also be used inprotein expression, including but not limited to, fermentors, shakeflasks, fluidized bed bioreactors, hollow fiber bioreactors, rollerbottle culture systems, and stirred tank bioreactor systems. Each ofthese methods can be performed in a batch, fed-batch, or continuous modeprocess.

Human ABP of the invention can generally be recovered using methodsstandard in the art. For example, culture medium or cell lysate can becentrifuged or filtered to remove cellular debris. The supernatant maybe concentrated or diluted to a desired volume or diafiltered into asuitable buffer to condition the preparation for further purification.Further purification of the ABP of the present invention includeseparating deamidated and clipped forms of the ABP variant from theintact form.

Any of the following exemplary procedures can be employed forpurification of antigen-binding polypeptides of the invention: affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;reverse phase HPLC; gel filtration (using, including but not limited to,SEPHADEX G-75); hydrophobic interaction chromatography; size-exclusionchromatography, metal-chelate chromatography;ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfateprecipitation; chromatofocusing; displacement chromatography;electrophoretic procedures (including but not limited to preparativeisoelectric focusing), differential solubility (including but notlimited to ammonium sulfate precipitation), SDS-PAGE, or extraction.

Proteins of the present invention, including but not limited to,proteins comprising unnatural amino acids, antibodies to proteinscomprising unnatural amino acids, binding partners for proteinscomprising unnatural amino acids, etc., can be purified, eitherpartially or substantially to homogeneity, according to standardprocedures known to and used by those of skill in the art. Accordingly,polypeptides of the invention can be recovered and purified by any of anumber of methods well known in the art, including but not limited to,ammonium sulfate or ethanol precipitation, acid or base extraction,column chromatography, affinity column chromatography, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, hydroxylapatite chromatography, lectinchromatography, gel electrophoresis and the like. Protein refoldingsteps can be used, as desired, in making correctly folded matureproteins. High performance liquid chromatography (HPLC), affinitychromatography or other suitable methods can be employed in finalpurification steps where high purity is desired. In one embodiment,antibodies made against unnatural amino acids (or proteins comprisingunnatural amino acids) are used as purification reagents, including butnot limited to, for affinity-based purification of proteins comprisingone or more unnatural amino acid(s). Once purified, partially or tohomogeneity, as desired, the polypeptides are optionally used for a widevariety of utilities, including but not limited to, as assay components,therapeutics, prophylaxis, diagnostics, research reagents, and/or asimmunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein folding methods are well known in the art,including, but not limited to, those set forth in R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982); Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc.N.Y. (1990); Sandana, (1997) Bioseparation of Proteins, Academic Press,Inc.; Bollag et al. (1996) Protein Methods, 2nd Edition Wiley-Liss, NY;Walker, (1996) The Protein Protocols Handbook Humana Press, NJ, Harrisand Angal, (1990) Protein Purification Applications: A PracticalApproach IRL Press at Oxford, Oxford, England; Harris and Angal, ProteinPurification Methods: A Practical Approach IRL Press at Oxford, Oxford,England; Scopes, (1993) Protein Purification Principles and Practice 3rdEdition Springer Verlag, NY; Janson and Ryden, (1998) ProteinPurification: Principles, High Resolution Methods and Applications,Second Edition Wiley-VCH, NY; and Walker (1998), Protein Protocols onCD-ROM Humana Press, NJ; and the references cited therein.

One advantage of producing a protein or polypeptide of interest with anunnatural amino acid in a eukaryotic host cell or non-eukaryotic hostcell is that typically the proteins or polypeptides will be folded intheir native conformations. However, in certain embodiments of theinvention, those of skill in the art will recognize that, aftersynthesis, expression and/or purification, proteins can possess aconformation different from the desired conformations of the relevantpolypeptides. In one aspect of the invention, the expressed protein isoptionally denatured and then renatured. This is accomplished utilizingmethods known in the art, including but not limited to, by adding achaperonin to the protein or polypeptide of interest, by solubilizingthe proteins in a chaotropic agent such as guanidine HCl, utilizingprotein disulfide isomerase, etc.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are well knownto those of skill in the art (see, the references above, and Debinski,et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan(1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992) Anal.Biochem., 205: 263-270). Debinski, et al., for example, describe thedenaturation and reduction of inclusion body proteins in guanidine-DTE.The proteins can be refolded in a redox buffer containing, including butnot limited to, oxidized glutathione and L-arginine. Refolding reagentscan be flowed or otherwise moved into contact with the one or morepolypeptide or other expression product, or vice-versa.

In the case of prokaryotic production of ABP, ABP thus produced may bemisfolded and thus lacks or has reduced biological activity. Thebioactivity of the protein may be restored by “refolding”. In general,misfolded ABP is refolded by solubilizing (where the ABP is alsoinsoluble), unfolding and reducing the polypeptide chain using, forexample, one or more chaotropic agents (e.g. urea and/or guanidine) anda reducing agent capable of reducing disulfide bonds (e.g.dithiothreitol, DTT or 2-mercaptoethanol, 2-ME). At a moderateconcentration of chaotrope, an oxidizing agent is then added (e.g.,oxygen, cystine or cystamine), which allows the reformation of disulfidebonds. ABP may be refolded using standard methods known in the art, suchas those described in U.S. Pat. Nos. 4,511,502, 4,511,503, and4,512,922, which are incorporated by reference herein. The ABP may alsobe cofolded with other proteins to form heterodimers or heteromultimers.After refolding or cofolding, the ABP is preferably further purified.

General Purification Methods Any one of a variety of isolation steps maybe performed on the cell lysate comprising ABP or on any ABP mixturesresulting from any isolation steps including, but not limited to,affinity chromatography, ion exchange chromatography, hydrophobicinteraction chromatography, gel filtration chromatography, highperformance liquid chromatography (“HPLC”), reversed phase-HPLC(“RP-HPLC”), expanded bed adsorption, or any combination and/orrepetition thereof and in any appropriate order.

Equipment and other necessary materials used in performing thetechniques described herein are commercially available. Pumps, fractioncollectors, monitors, recorders, and entire systems are available from,for example, Applied Biosystems (Foster City, Calif.), Bio-RadLaboratories, Inc. (Hercules, Calif.), and Amersham Biosciences, Inc.(Piscataway, N.J.). Chromatographic materials including, but not limitedto, exchange matrix materials, media, and buffers are also availablefrom such companies.

Equilibration, and other steps in the column chromatography processesdescribed herein such as washing and elution, may be more rapidlyaccomplished using specialized equipment such as a pump. Commerciallyavailable pumps include, but are not limited to, HILOAD® Pump P-50,Peristaltic Pump P-1, Pump P-901, and Pump P-903 (Amersham Biosciences,Piscataway, N.J.).

Examples of fraction collectors include RediFrac Fraction Collector,FRAC-100 and FRAC-200 Fraction Collectors, and SUPERFRAC® FractionCollector (Amersham Biosciences, Piscataway, N.J.). Mixers are alsoavailable to form pH and linear concentration gradients. Commerciallyavailable mixers include Gradient Mixer GM-1 and In-Line Mixers(Amersham Biosciences, Piscataway, N.J.).

The chromatographic process may be monitored using any commerciallyavailable monitor. Such monitors may be used to gather information likeUV, pH, and conductivity. Examples of detectors include Monitor UV-1,UVICORD® S II, Monitor UV-M II, Monitor UV-900, Monitor UPC-900, MonitorpH/C-900, and Conductivity Monitor (Amersham Biosciences, Piscataway,N.J.). Indeed, entire systems are commercially available including thevarious AKTA® systems from Amersham Biosciences (Piscataway, N.J.).

In one embodiment of the present invention, for example, the ABP may bereduced and denatured by first denaturing the resultant purified ABP inurea, followed by dilution into TRIS buffer containing a reducing agent(such as DTT) at a suitable pH. In another embodiment, the ABP isdenatured in urea in a concentration range of between about 2 M to about9 M, followed by dilution in TRIS buffer at a pH in the range of about5.0 to about 8.0. The refolding mixture of this embodiment may then beincubated. In one embodiment, the refolding mixture is incubated at roomtemperature for four to twenty-four hours. The reduced and denatured ABPmixture may then be further isolated or purified.

As stated herein, the pH of the first ABP mixture may be adjusted priorto performing any subsequent isolation steps. In addition, the first ABPmixture or any subsequent mixture thereof may be concentrated usingtechniques known in the art. Moreover, the elution buffer comprising thefirst ABP mixture or any subsequent mixture thereof may be exchanged fora buffer suitable for the next isolation step using techniques wellknown to those of ordinary skill in the art.

Ion Exchange Chromatography In one embodiment, and as an optional,additional step, ion exchange chromatography may be performed on thefirst ABP mixture. See generally ION EXCHANGE CHROMATOGRAPHY: PRINCIPLESAND METHODS (Cat. No. 18-1114-21, Amersham Biosciences (Piscataway,N.J.)). Commercially available ion exchange columns include HITRAP®,HIPREP®, and HILOAD® Columns (Amersham Biosciences, Piscataway, N.J.).Such columns utilize strong anion exchangers such as Q SEPHAROSE® FastFlow, Q SEPHAROSE® High Performance, and Q SEPHAROSE® XL; strong cationexchangers such as SP SEPHAROSE® High Performance, SP SEPHAROSE® FastFlow, and SP SEPHAROSE® XL; weak anion exchangers such as DEAESEPHAROSE® Fast Flow; and weak cation exchangers such as CM SEPHAROSE®Fast Flow (Amersham Biosciences, Piscataway, N.J.). Anion or cationexchange column chromatography may be performed on the ABP at any stageof the purification process to isolate substantially purified ABP. Thecation exchange chromatography step may be performed using any suitablecation exchange matrix. Useful cation exchange matrices include, but arenot limited to, fibrous, porous, non-porous, microgranular, beaded, orcross-linked cation exchange matrix materials. Such cation exchangematrix materials include, but are not limited to, cellulose, agarose,dextran, polyacrylate, polyvinyl, polystyrene, silica, polyether, orcomposites of any of the foregoing.

The cation exchange matrix may be any suitable cation exchangerincluding strong and weak cation exchangers. Strong cation exchangersmay remain ionized over a wide pH range and thus, may be capable ofbinding ABP over a wide pH range. Weak cation exchangers, however, maylose ionization as a function of pH. For example, a weak cationexchanger may lose charge when the pH drops below about pH 4 or pH 5.Suitable strong cation exchangers include, but are not limited to,charged functional groups such as sulfopropyl (SP), methyl sulfonate(S), or sulfoethyl (SE). The cation exchange matrix may be a strongcation exchanger, preferably having an ABP binding pH range of about 2.5to about 6.0. Alternatively, the strong cation exchanger may have an ABPbinding pH range of about pH 2.5 to about pH 5.5. The cation exchangematrix may be a strong cation exchanger having an ABP binding pH ofabout 3.0. Alternatively, the cation exchange matrix may be a strongcation exchanger, preferably having an ABP binding pH range of about 6.0to about 8.0. The cation exchange matrix may be a strong cationexchanger preferably having an ABP binding pH range of about 8.0 toabout 12.5. Alternatively, the strong cation exchanger may have an ABPbinding pH range of about pH 8.0 to about pH 12.0.

Prior to loading the ABP, the cation exchange matrix may beequilibrated, for example, using several column volumes of a dilute,weak acid, e.g., four column volumes of 20 mM acetic acid, pH 3.Following equilibration, the ABP may be added and the column may bewashed one to several times, prior to elution of substantially purifiedABP, also using a weak acid solution such as a weak acetic acid orphosphoric acid solution. For example, approximately 2-4 column volumesof 20 mM acetic acid, pH 3, may be used to wash the column. Additionalwashes using, e.g., 2-4 column volumes of 0.05 M sodium acetate, pH 5.5,or 0.05 M sodium acetate mixed with 0.1 M sodium chloride, pH 5.5, mayalso be used. Alternatively, using methods known in the art, the cationexchange matrix may be equilibrated using several column volumes of adilute, weak base.

Alternatively, substantially purified ABP may be eluted by contactingthe cation exchanger matrix with a buffer having a sufficiently low pHor ionic strength to displace the ABP from the matrix. The pH of theelution buffer may range from about pH 2.5 to about pH 6.0. Morespecifically, the pH of the elution buffer may range from about pH 2.5to about pH 5.5, about pH 2.5 to about pH 5.0. The elution buffer mayhave a pH of about 3.0. In addition, the quantity of elution buffer mayvary widely and will generally be in the range of about 2 to about 10column volumes. Moreover, suitable buffers known to those of skill inthe art may find use herein including, but not limited to, citrate,phosphate, formate, HEPES, and MES buffers ranging in concentration fromat least about 5 mM to at least about 100 mM.

Following adsorption of the ABP polypeptide to the cation exchangermatrix, substantially purified ABP polypeptide may be eluted bycontacting the matrix with a buffer having a sufficiently high pH orionic strength to displace the ABP from the matrix. Suitable buffers foruse in high pH elution of substantially purified ABP may include, butare not limited to, citrate, phosphate, formate, acetate, HEPES, and MESbuffers ranging in concentration from at least about 5 mM to at leastabout 100 mM.

Reverse-Phase Chromatography RP-HPLC may be performed to purify proteinsfollowing suitable protocols that are known to those of ordinary skillin the art. See, e.g., Pearson et al., ANAL BIOCHEM. (1982) 124:217-230(1982); Rivier et al., J. CHROM. (1983) 268:112-119; Kunitani et al., J.CHROM. (1986) 359:391-402. RP-HPLC may be performed on the ABP toisolate substantially purified ABP. In this regard, silica derivatizedresins with alkyl functionalities with a wide variety of lengths,including, but not limited to, at least about C₃ to at least about C₃₀,at least about C₃ to at least about C₂₀, or at least about C₃ to atleast about C₁₈, resins may be used. Alternatively, a polymeric resinmay be used. For example, TosoHaas Amberchrome CG1000sd resin may beused, which is a styrene polymer resin. Cyano or polymeric resins with awide variety of alkyl chain lengths may also be used. Furthermore, theRP-HPLC column may be washed with a solvent such as ethanol. The SourceRP column is another example of a RP-HPLC column.

A suitable elution buffer containing an ion pairing agent and an organicmodifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile orethanol, may be used to elute the ABP from the RP-HPLC column. The mostcommonly used ion pairing agents include, but are not limited to, aceticacid, formic acid, perchloric acid, phosphoric acid, trifluoroaceticacid, heptafluorobutyric acid, triethylamine, tetramethylammonium,tetrabutylammonium, and triethylammonium acetate. Elution may beperformed using one or more gradients or isocratic conditions, withgradient conditions preferred to reduce the separation time and todecrease peak width. Another method involves the use of two gradientswith different solvent concentration ranges. Examples of suitableelution buffers for use herein may include, but are not limited to,ammonium acetate and acetonitrile solutions.

Hydrophobic Interaction Chromatography Purification TechniquesHydrophobic interaction chromatography (HIC) may be performed on theABP. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK:PRINCIPLES AND METHODS (Cat. No. 18-1020-90, Amersham Biosciences(Piscataway, N.J.) which is incorporated by reference herein. SuitableHIC matrices may include, but are not limited to, alkyl- oraryl-substituted matrices, such as butyl-, hexyl-, octyl- orphenyl-substituted matrices including agarose, cross-linked agarose,sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate)matrices, and mixed mode resins, including but not limited to, apolyethyleneamine resin or a butyl- or phenyl-substitutedpoly(methacrylate) matrix. Commercially available sources forhydrophobic interaction column chromatography include, but are notlimited to, HITRAP®, HIPREP®, and HILOAD® columns (Amersham Biosciences,Piscataway, N.J.).

Briefly, prior to loading, the HIC column may be equilibrated usingstandard buffers known to those of ordinary skill in the art, such as anacetic acid/sodium chloride solution or HEPES containing ammoniumsulfate. Ammonium sulfate may be used as the buffer for loading the HICcolumn. After loading the ABP, the column may then washed using standardbuffers and conditions to remove unwanted materials but retaining theABP on the HIC column. ABP may be eluted with about 3 to about 10 columnvolumes of a standard buffer, such as a HEPES buffer containing EDTA andlower ammonium sulfate concentration than the equilibrating buffer, oran acetic acid/sodium chloride buffer, among others. A decreasing linearsalt gradient using, for example, a gradient of potassium phosphate, mayalso be used to elute the ABP molecules. The eluant may then beconcentrated, for example, by filtration such as diafiltration orultrafiltration. Diafiltration may be utilized to remove the salt usedto elute ABP.

Other Purification Techniques Yet another isolation step using, forexample, gel filtration (GEL FILTRATION: PRINCIPLES AND METHODS (Cat.No. 18-1022-18, Amersham Biosciences, Piscataway, N.J.) which isincorporated by reference herein, hydroxyapatite chromatography(suitable matrices include, but are not limited to, HA-Ultrogel, HighResolution (Calbiochem), CHT Ceramic Hydroxyapatite (BioRad), Bio-GelHTP Hydroxyapatite (BioRad)), HPLC, expanded bed adsorption,ultrafiltration, diafiltration, lyophilization, and the like, may beperformed on the first ABP mixture or any subsequent mixture thereof, toremove any excess salts and to replace the buffer with a suitable bufferfor the next isolation step or even formulation of the final drugproduct.

The non-naturally encoded amino acid present in the ABP may also beutilized to provide separation from other cellular proteins that do notcontain the non-naturally encoded amino acid. Since the non-naturallyencoded amino acid may comprise unique chemical functional groups, thecoupling of the unique functional group to another molecule may providea substantial purification step. For example, the non-naturally encodedamino acid may be coupled to another molecule that facilitatesseparation from other proteins. Such molecules for coupling to thenon-natural amino acid include, but are not limited to, PEG and otherpolymers, beads, and other solid substances.

The yield of ABP, including substantially purified ABP, may be monitoredat each step described herein using techniques known to those ofordinary skill in the art. Such techniques may also be used to assessthe yield of substantially purified ABP following the last isolationstep. For example, the yield of ABP may be monitored using any ofseveral reverse phase high pressure liquid chromatography columns,having a variety of alkyl chain lengths such as cyano RP-HPLC,C₁₈RP-HPLC; as well as cation exchange HPLC and gel filtration HPLC.

In specific embodiments of the present invention, the yield of ABP aftereach purification step may be at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, at least about 99.9%, or atleast about 99.99%, of the ABP in the starting material for eachpurification step.

Purity may be determined using standard techniques, such as SDS-PAGE, orby measuring ABP using Western blot and ELISA assays. For example,polyclonal antibodies may be generated against proteins isolated fromnegative control yeast fermentation and the cation exchange recovery.The antibodies may also be used to probe for the presence ofcontaminating host cell proteins.

RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, thesurfaces of which carry C4-alkyl chains. The separation of ABP from theproteinaceous impurities is based on differences in the strength ofhydrophobic interactions. Elution is performed with an acetonitrilegradient in diluted trifluoroacetic acid. Preparative HPLC is performedusing a stainless steel column (filled with 2.8 to 3.2 liter of Vydac C4silicagel). The Hydroxyapatite Ultrogel eluate is acidified by addingtrifluoroacetic acid and loaded onto the Vydac C4 column. For washingand elution an acetonitrile gradient in diluted trifluoroacetic acid isused. Fractions are collected and immediately neutralized with phosphatebuffer. The ABP fractions which are within the IPC limits are pooled.

DEAE Sepharose (Pharmacia) material consists of diethylaminoethyl(DEAE)-groups which are covalently bound to the surface of Sepharosebeads. The binding of ABP to the DEAE groups is mediated by ionicinteractions. Acetonitrile and trifluoroacetic acid pass through thecolumn without being retained. After these substances have been washedoff, trace impurities are removed by washing the column with acetatebuffer at a low pH. Then the column is washed with neutral phosphatebuffer and ABP is eluted with a buffer with increased ionic strength.The column is packed with DEAE Sepharose fast flow. The column volume isadjusted to assure a ABP load in the range of 3-10 mg ABP polypeptide/mlgel. The column is washed with water and equilibration buffer(sodium/potassium phosphate). The pooled fractions of the HPLC eluateare loaded and the column is washed with equilibration buffer. Then thecolumn is washed with washing buffer (sodium acetate buffer) followed bywashing with equilibration buffer. Subsequently, ABP is eluted from thecolumn with elution buffer (sodium chloride, sodium/potassium phosphate)and collected in a single fraction in accordance with the master elutionprofile. The eluate of the DEAE Sepharose column is adjusted to thespecified conductivity. The resulting drug substance is sterile filteredinto Teflon bottles and stored at −70° C.

Additional methods that may be employed include, but are not limited to,steps to remove endotoxins. Endotoxins are lipopoly-saccharides (LPSs)which are located on the outer membrane of Gram-negative host cells,such as, for example, Escherichia coli. Methods for reducing endotoxinlevels are known to one skilled in the art and include, but are notlimited to, purification techniques using silica supports, glass powderor hydroxyapatite, reverse-phase, affinity, size-exclusion,anion-exchange chromatography, hydrophobic interaction chromatography, acombination of these methods, and the like. Modifications or additionalmethods may be required to remove contaminants such as co-migratingproteins from the polypeptide of interest

A wide variety of methods and procedures can be used to assess the yieldand purity of a ABP protein one or more non-naturally encoded aminoacids, including but not limited to, the Bradford assay, SDS-PAGE,silver stained SDS-PAGE, coomassie stained SDS-PAGE, mass spectrometry(including but not limited to, MALDI-TOF) and other methods forcharacterizing proteins known to one skilled in the art.

VII. Expression in Alternate Systems

Several strategies have been employed to introduce unnatural amino acidsinto proteins in non-recombinant host cells, mutagenized host cells, orin cell-free systems. These systems are also suitable for use in makingthe antigen-binding polypeptides of the present invention.Derivatization of amino acids with reactive side-chains such as Lys, Cysand Tyr resulted in the conversion of lysine to N²-acetyl-lysine.Chemical synthesis also provides a straightforward method to incorporateunnatural amino acids. With the recent development of enzymatic ligationand native chemical ligation of peptide fragments, it is possible tomake larger proteins. See, e.g., P. E. Dawson and S. B. H. Kent, Annu.Rev. Biochem., 69:923 (2000). A general in vitro biosynthetic method inwhich a suppressor tRNA chemically acylated with the desired unnaturalamino acid is added to an in vitro extract capable of supporting proteinbiosynthesis, has been used to site-specifically incorporate over 100unnatural amino acids into a variety of proteins of virtually any size.See, e.g., V. W. Cornish, D. Mendel and P. G. Schultz, Angew. Chem. Int.Ed. Engl., 1995, 34:621 (1995); C. J. Noren, S. J. Anthony-Cahill, M. C.Griffith, P. G. Schultz, A general method for site-specificincorporation of unnatural amino acids into proteins, Science244:182-188 (1989); and, J. D. Bain, C. G. Glabe, T. A. Dix, A. R.Chamberlin, E. S. Diala, Biosynthetic site-specific incorporation of anon-natural amino acid into a polypeptide, J. Am. Chem. Soc.111:8013-8014 (1989). A broad range of functional groups has beenintroduced into proteins for studies of protein stability, proteinfolding, enzyme mechanism, and signal transduction.

An in vivo method, termed selective pressure incorporation, wasdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophic strain, inwhich the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been incorporated in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Karnbrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind,L. Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionineanalogs with alkene or alkyne functionalities have also beenincorporated efficiently, allowing for additional modification ofproteins by chemical means. See, e.g., J. C. M. van Hest and D. A.Tirrell, FEBS Lett., 428:68 (1998); J. C. M. van Hest, K. L. Kiick andD. A. Tirrell, J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick andD. A. Tirrell, Tetrahedron, 56:9487 (2000); U.S. Pat. No. 6,586,207;U.S. Patent Publication 2002/0042097, which are incorporated byreference herein.

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. For example, replacement of Ala²⁹⁴ by Gly inEscherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the sizeof substrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS. Nishimura, J. Biol. Chem., 275:40324 (2000).

Another strategy to incorporate unnatural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAValwith Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys (—SH group of Cys is replaced with —CH3 inAbu), the mutant ValRS also incorporates Abu into proteins when thismutant Escherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing novel amino acids. Forexample, see the following publications and references cited within,which are as follows: Crick, F. J. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides.XXVI. The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am. Chem,88(24):5914-5919 (1966); Kaiser, E. T. Synthetic approaches tobiologically active peptides and proteins including enzymes, Acc ChemRes, 47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptidesegment coupling catalyzed by the semisynthetic enzyme thiosubtilisin, JAm Chem Soc, 3808-3810 (1987); Schnolzer, M., Kent, S B H. Constructingproteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256(5054):221-225 (1992);Chaiken, I. M. Semisynthetic peptides and proteins, CRC Crit. RevBiochem, 11(3):255-301 (1981); Offord, R. E. Protein engineering bychemical means? Protein Eng., 1(3):151-157 (1987); and, Jackson, D. Y.,Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J. A. A DesignedPeptide Ligase for Total Synthesis of Ribonuclease A with UnnaturalCatalytic Residues, Science, 266(5183):243 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation ofa hybrid sequence-specific single-stranded deoxyribonuclease, Science,238(4832):1401-1403 (1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E.The chemical modification of enzymatic specificity, Annu Rev Biochem,54:565-595 (1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation ofenzyme active sites, Science, 226(4674):505-511 (1984); Neet, K. E.,Nanci A, Koshland, D. E. Properties of thiol-subtilisin, J. Biol. Chem.,243(24):6392-6401 (1968); Polgar, L. B., M. L. A new enzyme containing asynthetically formed active site. Thiol-subtilisin. J. Am. Chem Soc,3153-3154 (1966); and, Pollack, S. J., Nakayama, G. Schultz, P. G.Introduction of nucleophiles and spectroscopic probes into antibodycombining sites, Science, 242(4881): 1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U. C.,Walter, P., Hohnson, A. E. Photocrosslinking of the signal sequence ofnascent preprolactin of the 54-kilodalton polypeptide of the signalrecognition particle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).

Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid.See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G.A general method for site-specific incorporation of unnatural aminoacids into proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am. Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., 301-336 (1992); and,Mendel, D., Cornish, V. W. & Schultz, P. G. Site-Directed Mutagenesiswith an Expanded Genetic Code, Annu Rev Biophys. Biomol Struct. 24,435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an unnatural amino acid.Conventional site-directed mutagenesis was used to introduce the stopcodon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′, 3′ Exonuclease inphosphorothioate-based oligonucleotide-directed mutagenesis, NucleicAcids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the unnatural amino acid was incorporated in response to the UAGcodon which gave a protein containing that amino acid at the specifiedposition. Experiments using [³H]-Phe and experiments with α-hydroxyacids demonstrated that only the desired amino acid is incorporated atthe position specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432;and, Ellman, J. A., Mendel, D., Schultz, P. G. Site-specificincorporation of novel backbone structures into proteins, Science,255(5041):197-200 (1992).

Microinjection techniques have also been use incorporate unnatural aminoacids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired unnatural amino acid. Thetranslational machinery of the oocyte then inserts the unnatural aminoacid at the position specified by UAG. This method has allowed in vivostructure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of alpha hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms. See, e.g., P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers the advantages of high yields of mutant proteins,technical ease, the potential to study the mutant proteins in cells orpossibly in living organisms and the use of these mutant proteins intherapeutic treatments. The ability to include unnatural amino acidswith various sizes, acidities, nucleophilicities, hydrophobicities, andother properties into proteins can greatly expand our ability torationally and systematically manipulate the structures of proteins,both to probe protein function and create new proteins or organisms withnovel properties. However, the process is difficult, because the complexnature of tRNA-synthetase interactions that are required to achieve ahigh degree of fidelity in protein translation.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci., 7:419 (1998).

It may also be possible to obtain expression of an ABP polynucleotide ofthe present invention using a cell-free (in-vitro) translational system.In these systems, which can include either mRNA as a template (in-vitrotranslation) or DNA as a template (combined in-vitro transcription andtranslation), the in vitro synthesis is directed by the ribosomes.Considerable effort has been applied to the development of cell-freeprotein expression systems. See, e.g., Kim, D.-M. and J. R. Swartz,Biotechnology and Bioengineering, 74 :309-316 (2001); Kim, D.-M. and J.R. Swartz, Biotechnology Letters, 22, 1537-1542, (2000); Kim, D.-M., andJ. R. Swartz, Biotechnology Progress, 16, 385-390, (2000); Kim, D.-M.,and J. R. Swartz, Biotechnology and Bioengineering, 66, 180-188, (1999);and Patnaik, R. and J. R. Swartz, Biotechniques 24, 862-868, (1998);U.S. Pat. No. 6,337,191; U.S. Patent Publication No. 2002/0081660; WO00/55353; WO 90/05785, which are incorporated by reference herein.Another approach that may be applied to the expression ofantigen-binding polypeptides comprising a non-naturally encoded aminoacid includes the mRNA-peptide fusion technique. See, e.g., R. Robertsand J. Szostak, Proc. Natl. Acad. Sci. (USA) 94:12297-12302 (1997); A.Frankel, et al., Chemistry & Biology 10:1043-1050 (2003). In thisapproach, an mRNA template linked to puromycin is translated intopeptide on the ribosome. If one or more tRNA molecules has beenmodified, non-natural amino acids can be incorporated into the peptideas well. After the last mRNA codon has been read, puromycin captures theC-terminus of the peptide. If the resulting mRNA-peptide conjugate isfound to have interesting properties in an in vitro assay, its identitycan be easily revealed from the mRNA sequence. In this way, one mayscreen libraries of antigen-binding polypeptides comprising one or morenon-naturally encoded amino acids to identify polypeptides havingdesired properties. More recently, in vitro ribosome translations withpurified components have been reported that permit the synthesis ofpeptides substituted with non-naturally encoded amino acids. See, e.g.,A. Forster et al., Proc. Natl. Acad. Sci. (USA) 100:6353 (2003).

IX. Macromolecular Polymers Coupled to ABP

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide, including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a water-soluble dendrimer; a cyclodextrin; aninhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spinlabel; a fluorophore, a metal-containing moiety; a radioactive moiety; anovel functional group; a group that covalently or noncovalentlyinteracts with other molecules; a photocaged moiety; a photoisomerizablemoiety; biotin; a derivative of biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; a small molecule; or anycombination of the above, or any other desirable compound or substance.As an illustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the following descriptionwill focus on adding macromolecular polymers to the non-natural aminoacid polypeptide with the understanding that the compositions, methods,techniques and strategies described thereto are also applicable (withappropriate modifications, if necessary and for which one of skill inthe art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can belinked to antigen-binding polypeptides of the present invention tomodulate biological properties of the ABP, and/or provide new biologicalproperties to the ABP molecule. These macromolecular polymers can belinked to the ABP via a naturally encoded amino acid, via anon-naturally encoded amino acid, or any functional substituent of anatural or non-natural amino acid, or any substituent or functionalgroup added to a natural or non-natural amino acid. The molecular weightof the polymer may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more.

The present invention provides substantially homogenous preparations ofpolymer:protein conjugates. “Substantially homogenous” as used hereinmeans that polymer:protein conjugate molecules are observed to begreater than half of the total protein. The polymer:protein conjugatehas biological activity and the present “substantially homogenous”PEGylated ABP preparations provided herein are those which arehomogenous enough to display the advantages of a homogenous preparation,e.g., ease in clinical application in predictability of lot to lotpharmacokinetics.

One may also choose to prepare a mixture of polymer:protein conjugatemolecules, and the advantage provided herein is that one may select theproportion of mono-polymer:protein conjugate to include in the mixture.Thus, if desired, one may prepare a mixture of various proteins withvarious numbers of polymer moieties attached (i.e., di-, tri-, tetra-,etc.) and combine said conjugates with the mono-polymer:proteinconjugate prepared using the methods of the present invention, and havea mixture with a predetermined proportion of mono-polymer:proteinconjugates.

The polymer selected may be water soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be branched or unbranched.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable.

The proportion of polyethylene glycol molecules to protein moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

As used herein, and when contemplating PEG:ABP conjugates, the term“therapeutically effective amount” refers to an amount which gives thedesired benefit to a patient. The amount will vary from one individualto another and will depend upon a number of factors, including theoverall physical condition of the patient and the underlying cause ofthe condition to be treated. The amount of ABP polypeptide used fortherapy gives an acceptable rate of change and maintains desiredresponse at a beneficial level. A therapeutically effective amount ofthe present compositions may be readily ascertained by one skilled inthe art using publicly available materials and procedures.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water solublepolymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG),but other water soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments of this invention.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented as linked to the ABP by the formula:XO—(CH₂CH₂O)_(n)—CH₂CH₂—Ywhere n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl.

In some cases, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Alternatively,the PEG can terminate with a reactive group, thereby forming abifunctional polymer. Typical reactive groups can include those reactivegroups that are commonly used to react with the functional groups foundin the 20 common amino acids (including but not limited to, maleimidegroups, activated carbonates (including but not limited to,p-nitrophenyl ester), activated esters (including but not limited to,N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well asfunctional groups that are inert to the 20 common amino acids but thatreact specifically with complementary functional groups present innon-naturally encoded amino acids (including but not limited to, azidegroups, alkyne groups). It is noted that the other end of the PEG, whichis shown in the above formula by Y, will attach either directly orindirectly to an antigen-binding polypeptide via a naturally-occurringor non-naturally encoded amino acid. For instance, Y may be an amide,carbamate or urea linkage to an amine group (including but not limitedto, the epsilon amine of lysine or the N-terminus) of the polypeptide.Alternatively, Y may be a maleimide linkage to a thiol group (includingbut not limited to, the thiol group of cysteine). Alternatively, Y maybe a linkage to a residue not commonly accessible via the 20 commonamino acids. For example, an azide group on the PEG can be reacted withan alkyne group on the ABP to form a Huisgen [3+2]cycloaddition product.Alternatively, an alkyne group on the PEG can be reacted with an azidegroup present in a non-naturally encoded amino acid to form a similarproduct. In some embodiments, a strong nucleophile (including but notlimited to, hydrazine, hydrazide, hydroxylamine, semicarbazide) can bereacted with an aldehyde or ketone group present in a non-naturallyencoded amino acid to form a hydrazone, oxime or semicarbazone, asapplicable, which in some cases can be further reduced by treatment withan appropriate reducing agent. Alternatively, the strong nucleophile canbe incorporated into the ABP via a non-naturally encoded amino acid andused to react preferentially with a ketone or aldehyde group present inthe water soluble polymer.

Any molecular mass for a PEG can be used as practically desired,including but not limited to, from about 100 Daltons (Da) to 100,000 Daor more as desired (including but not limited to, sometimes 0.1-50 kDaor 10-40 kDa). Branched chain PEGs, including but not limited to, PEGmolecules with each chain having a MW ranging from 1-100 kDa (includingbut not limited to, 1-50 kDa or 5-20 kDa) can also be used. A wide rangeof PEG molecules are described in, including but not limited to, theShearwater Polymers, Inc. catalog, Nektar Therapeutics catalog,incorporated herein by reference.

Generally, at least one terminus of the PEG molecule is available forreaction with the non-naturally-encoded amino acid. For example, PEGderivatives bearing alkyne and azide moieties for reaction with aminoacid side chains can be used to attach PEG to non-naturally encodedamino acids as described herein. If the non-naturally encoded amino acidcomprises an azide, then the PEG will typically contain either an alkynemoiety to effect formation of the [3+2]cycloaddition product or anactivated PEG species (i.e., ester, carbonate) containing a phosphinegroup to effect formation of the amide linkage. Alternatively, if thenon-naturally encoded amino acid comprises an alkyne, then the PEG willtypically contain an azide moiety to effect formation of the [3+2]Huisgen cycloaddition product. If the non-naturally encoded amino acidcomprises a carbonyl group, the PEG will typically comprise a potentnucleophile (including but not limited to, a hydrazide, hydrazine,hydroxylamine, or semicarbazide functionality) in order to effectformation of corresponding hydrazone, oxime, and semicarbazone linkages,respectively. In other alternatives, a reverse of the orientation of thereactive groups described above can be used, i.e., an azide moiety inthe non-naturally encoded amino acid can be reacted with a PEGderivative containing an alkyne.

In some embodiments, the ABP variant with a PEG derivative contains achemical functionality that is reactive with the chemical functionalitypresent on the side chain of the non-naturally encoded amino acid.

The invention provides in some embodiments azide- andacetylene-containing polymer derivatives comprising a water solublepolymer backbone having an average molecular weight from about 800 Da toabout 100,000 Da. The polymer backbone of the water-soluble polymer canbe poly(ethylene glycol). However, it should be understood that a widevariety of water soluble polymers including but not limited topoly(ethylene)glycol and other related polymers, including poly(dextran)and poly(propylene glycol), are also suitable for use in the practice ofthis invention and that the use of the term PEG or poly(ethylene glycol)is intended to encompass and include all such molecules. The term PEGincludes, but is not limited to, poly(ethylene glycol) in any of itsforms, including bifunctional PEG, multiarmed PEG, derivatized PEG,forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymershaving one or more functional groups pendent to the polymer backbone),or PEG with degradable linkages therein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about3 to about 4000, typically from about 20 to about 2000, is suitable foruse in the present invention. PEG having a molecular weight of fromabout 800 Da to about 100,000 Da are in some embodiments of the presentinvention particularly useful as the polymer backbone.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:-PEG-CO₂-PEG-+H₂O→PEG-CO₂H+HO-PEG-It is understood by those skilled in the art that the term poly(ethyleneglycol) or PEG represents or includes all the forms known in the artincluding but not limited to those disclosed herein.

Many other polymers are also suitable for use in the present invention.In some embodiments, polymer backbones that are water-soluble, with from2 to about 300 termini, are particularly useful in the invention.Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers thereof (including but not limited to copolymers of ethyleneglycol and propylene glycol), terpolymers thereof, mixtures thereof, andthe like. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 800 Da toabout 100,000 Da, often from about 6,000 Da to about 80,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated as being suitable for use inthe present invention.

In some embodiments of the present invention the polymer derivatives are“multi-functional”, meaning that the polymer backbone has at least twotermini, and possibly as many as about 300 termini, functionalized oractivated with a functional group. Multifunctional polymer derivativesinclude, but are not limited to, linear polymers having two termini,each terminus being bonded to a functional group which may be the sameor different.

In one embodiment, the polymer derivative has the structure:X-A-POLY-B-N═N═Nwherein:N═N═N is an azide moiety;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and morepreferably between 1-10 carbon atoms. A heteroatom such as nitrogen,oxygen or sulfur may be included with the alkyl chain. The alkyl chainmay also be branched at a heteroatom. Other examples of a linking moietyfor A and B include, but are not limited to, a multiply functionalizedaryl group, containing up to 10 and more preferably 5-6 carbon atoms.The aryl group may be substituted with one more carbon atoms, nitrogen,oxygen or sulfur atoms. Other examples of suitable linking groupsinclude those linking groups described in U.S. Pat. Nos. 5,932,462;5,643,575; and U.S. Pat. Appl. Publication 2003/0143596, each of whichis incorporated by reference herein. Those of ordinary skill in the artwill recognize that the foregoing list for linking moieties is by nomeans exhaustive and is merely illustrative, and that all linkingmoieties having the qualities described above are contemplated to besuitable for use in the present invention.

Examples of suitable functional groups for use as X include, but are notlimited to, hydroxyl, protected hydroxyl, alkoxyl, active ester, such asN-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, activecarbonate, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolylcarbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, aminooxy, protectedamine, hydrazide, protected hydrazide, protected thiol, carboxylic acid,protected carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxals, diones, mesylates, tosylates, tresylate, alkene, ketone, andazide. As is understood by those skilled in the art, the selected Xmoiety should be compatible with the azide group so that reaction withthe azide group does not occur. The azide-containing polymer derivativesmay be homobifunctional, meaning that the second functional group (i.e.,X) is also an azide moiety, or heterobifunctional, meaning that thesecond functional group is a different functional group.

The term “protected” refers to the presence of a protecting group ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the present invention.

Specific examples of terminal functional groups in the literatureinclude, but are not limited to, N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al.Makromol. Chem. 182:1379 (1981), Zaplipsky et al. Eur. Polym. J. 19:1177(1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301(1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zaplipsky Eds., ACS, Washington,D.C., 1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate(See, e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al. Macrolol. Chem. 180:1381 (1979), succinimidyl ester (see,e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S.Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J.Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354(1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Bio/Technology 8:343(1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of theabove references and patents are incorporated herein by reference.

In certain embodiments of the present invention, the polymer derivativesof the invention comprise a polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—N═N═Nwherein:X is a functional group as described above; andn is about 20 to about 4000.In another embodiment, the polymer derivatives of the invention comprisea polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—W—N═N═Nwherein:W is an aliphatic or aromatic linker moiety comprising between 1-10carbon atoms;n is about 20 to about 4000; andX is a functional group as described above. m is between 1 and 10.

The azide-containing PEG derivatives of the invention can be prepared bya variety of methods known in the art and/or disclosed herein. In onemethod, shown below, a water soluble polymer backbone having an averagemolecular weight from about 800 Da to about 100,000 Da, the polymerbackbone having a first terminus bonded to a first functional group anda second terminus bonded to a suitable leaving group, is reacted with anazide anion (which may be paired with any of a number of suitablecounter-ions, including sodium, potassium, tert-butylammonium and soforth). The leaving group undergoes a nucleophilic displacement and isreplaced by the azide moiety, affording the desired azide-containing PEGpolymer.X-PEG-L+N₃ ⁻→X-PEG-N₃

As shown, a suitable polymer backbone for use in the present inventionhas the formula X-PEG-L, wherein PEG is poly(ethylene glycol) and X is afunctional group which does not react with azide groups and L is asuitable leaving group. Examples of suitable functional groups include,but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl,amine, aminooxy, protected amine, protected hydrazide, protected thiol,carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine,and vinylpyridine, and ketone. Examples of suitable leaving groupsinclude, but are not limited to, chloride, bromide, iodide, mesylate,tresylate, and tosylate.

In another method for preparation of the azide-containing polymerderivatives of the present invention, a linking agent bearing an azidefunctionality is contacted with a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da,wherein the linking agent bears a chemical functionality that will reactselectively with a chemical functionality on the PEG polymer, to form anazide-containing polymer derivative product wherein the azide isseparated from the polymer backbone by a linking group.

An exemplary reaction scheme is shown below:X-PEG-M+N-linker-N═N═N→PG-X-PEG-linker-N═N═Nwherein:PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; andM is a functional group that is not reactive with the azidefunctionality but that will react efficiently and selectively with the Nfunctional group.

Examples of suitable functional groups include, but are not limited to,M being a carboxylic acid, carbonate or active ester if N is an amine; Mbeing a ketone if N is a hydrazide or aminooxy moiety; M being a leavinggroup if N is a nucleophile.

Purification of the crude product may be accomplished by known methodsincluding, but are not limited to, precipitation of the product followedby chromatography, if necessary.

A more specific example is shown below in the case of PEG diamine, inwhich one of the amines is protected by a protecting group moiety suchas tert-butyl-Boc and the resulting mono-protected PEG diamine isreacted with a linking moiety that bears the azide functionality:BocHN-PEG-NH₂+HO₂C—(CH₂)₃—N═N═N

In this instance, the amine group can be coupled to the carboxylic acidgroup using a variety of activating agents such as thionyl chloride orcarbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazoleto create an amide bond between the monoamine PEG derivative and theazide-bearing linker moiety. After successful formation of the amidebond, the resulting N-tert-butyl-Boc-protected azide-containingderivative can be used directly to modify bioactive molecules or it canbe further elaborated to install other useful functional groups. Forinstance, the N-t-Boc group can be hydrolyzed by treatment with strongacid to generate an omega-amino-PEG-azide. The resulting amine can beused as a synthetic handle to install other useful functionality such asmaleimide groups, activated disulfides, activated esters and so forthfor the creation of valuable heterobifunctional reagents.

Heterobifunctional derivatives are particularly useful when it isdesired to attach different molecules to each terminus of the polymer.For example, the omega-N-amino-N-azido PEG would allow the attachment ofa molecule having an activated electrophilic group, such as an aldehyde,ketone, activated ester, activated carbonate and so forth, to oneterminus of the PEG and a molecule having an acetylene group to theother terminus of the PEG.

In another embodiment of the invention, the polymer derivative has thestructure:X-A-POLY-B—C≡C—Rwherein:R can be either H or an alkyl, alkene, alkyoxy, or aryl or substitutedaryl group;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.

Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and morepreferably between 1-10 carbon atoms. A heteroatom such as nitrogen,oxygen or sulfur may be included with the alkyl chain. The alkyl chainmay also be branched at a heteroatom. Other examples of a linking moietyfor A and B include, but are not limited to, a multiply functionalizedaryl group, containing up to 10 and more preferably 5-6 carbon atoms.The aryl group may be substituted with one more carbon atoms, nitrogen,oxygen, or sulfur atoms. Other examples of suitable linking groupsinclude those linking groups described in U.S. Pat. Nos. 5,932,462 and5,643,575 and U.S. Pat. Appl. Publication 2003/0143596, each of which isincorporated by reference herein. Those of ordinary skill in the artwill recognize that the foregoing list for linking moieties is by nomeans exhaustive and is intended to be merely illustrative, and that awide variety of linking moieties having the qualities described aboveare contemplated to be useful in the present invention.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidylesters and 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, ketone, and acetylene. Aswould be understood, the selected X moiety should be compatible with theacetylene group so that reaction with the acetylene group does notoccur. The acetylene-containing polymer derivatives may behomobifunctional, meaning that the second functional group (i.e., X) isalso an acetylene moiety, or heterobifunctional, meaning that the secondfunctional group is a different functional group.

In another embodiment of the present invention, the polymer derivativescomprise a polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—C≡CHwherein:X is a functional group as described above;n is about 20 to about 4000; andm is between 1 and 10.Specific examples of each of the heterobifunctional PEG polymers areshown below.

The acetylene-containing PEG derivatives of the invention can beprepared using methods known to those skilled in the art and/ordisclosed herein. In one method, a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da, thepolymer backbone having a first terminus bonded to a first functionalgroup and a second terminus bonded to a suitable nucleophilic group, isreacted with a compound that bears both an acetylene functionality and aleaving group that is suitable for reaction with the nucleophilic groupon the PEG. When the PEG polymer bearing the nucleophilic moiety and themolecule bearing the leaving group are combined, the leaving groupundergoes a nucleophilic displacement and is replaced by thenucleophilic moiety, affording the desired acetylene-containing polymer.X-PEG-Nu+L-A-C→X-PEG-Nu-A-C≡CR′

As shown, a preferred polymer backbone for use in the reaction has theformula X-PEG-Nu, wherein PEG is poly(ethylene glycol), Nu is anucleophilic moiety and X is a functional group that does not react withNu, L or the acetylene functionality.

Examples of Nu include, but are not limited to, amine, alkoxy, aryloxy,sulfhydryl, imino, carboxylate, hydrazide, aminoxy groups that wouldreact primarily via a SN2-type mechanism. Additional examples of Nugroups include those functional groups that would react primarily via annucleophilic addition reaction. Examples of L groups include chloride,bromide, iodide, mesylate, tresylate, and tosylate and other groupsexpected to undergo nucleophilic displacement as well as ketones,aldehydes, thioesters, olefins, alpha-beta unsaturated carbonyl groups,carbonates and other electrophilic groups expected to undergo additionby nucleophiles.

In another embodiment of the present invention, A is an aliphatic linkerof between 1-10 carbon atoms or a substituted aryl ring of between 6-14carbon atoms. X is a functional group which does not react with azidegroups and L is a suitable leaving group

In another method for preparation of the acetylene-containing polymerderivatives of the invention, a PEG polymer having an average molecularweight from about 800 Da to about 100,000 Da, bearing either a protectedfunctional group or a capping agent at one terminus and a suitableleaving group at the other terminus is contacted by an acetylene anion.

An exemplary reaction scheme is shown below:X-PEG-L+—C≡CR′→X-PEG-C≡CR′wherein:PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; andR′ is either H, an alkyl, alkoxy, aryl or aryloxy group or a substitutedalkyl, alkoxyl, aryl or aryloxy group.

In the example above, the leaving group L should be sufficientlyreactive to undergo SN2-type displacement when contacted with asufficient concentration of the acetylene anion. The reaction conditionsrequired to accomplish SN2 displacement of leaving groups by acetyleneanions are well known in the art.

Purification of the crude product can usually be accomplished by methodsknown in the art including, but are not limited to, precipitation of theproduct followed by chromatography, if necessary.

Water soluble polymers can be linked to the antigen-binding polypeptidesof the invention. The water soluble polymers may be linked via anon-naturally encoded amino acid incorporated in the antigen-bindingpolypeptide or any functional group or substituent of a non-naturallyencoded or naturally encoded amino acid, or any functional group orsubstituent added to a non-naturally encoded or naturally encoded aminoacid. Alternatively, the water soluble polymers are linked to anantigen-binding polypeptide incorporating a non-naturally encoded aminoacid via a naturally-occurring amino acid (including but not limited to,cysteine, lysine or the amine group of the N-terminal residue). In somecases, the ABP of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10non-natural amino acids, wherein one or more non-naturally-encoded aminoacid(s) are linked to water soluble polymer(s) (including but notlimited to, PEG and/or oligosaccharides). In some cases, the ABP of theinvention further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morenaturally-encoded amino acid(s) linked to water soluble polymers. Insome cases, the ABP of the invention comprise one or more non-naturallyencoded amino acid(s) linked to water soluble polymers and one or morenaturally-occurring amino acids linked to water soluble polymers. Insome embodiments, the water soluble polymers used in the presentinvention enhance the serum half-life of the ABP relative to theunconjugated form.

The number of water soluble polymers linked to an antigen-bindingpolypeptide (i.e., the extent of PEGylation or glycosylation) of thepresent invention can be adjusted to provide an altered (including butnot limited to, increased or decreased) pharmacologic, pharmacokineticor pharmacodynamic characteristic such as in vivo half-life. In someembodiments, the half-life of ABP is increased at least about 10, 20,30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold, 10-fold, 50-fold, orat least about 100-fold over an unmodified polypeptide.

Peg Derivatives Containing a Strong Nucleophilic Group (i.e., Hydrazide,Hydrazine, Hydroxylamine or Semicarbazide)

In one embodiment of the present invention, an antigen-bindingpolypeptide comprising a carbonyl-containing non-naturally encoded aminoacid is modified with a PEG derivative that contains a terminalhydrazine, hydroxylamine, hydrazide or semicarbazide moiety that islinked directly to the PEG backbone.

In some embodiments, the hydroxylamine-terminal PEG derivative will havethe structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—O—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivative will have the structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—X—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the semicarbazide-containing PEG derivative willhave the structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, an antigen-binding polypeptidecomprising a carbonyl-containing amino acid is modified with a PEGderivative that contains a terminal hydroxylamine, hydrazide, hydrazine,or semicarbazide moiety that is linked to the PEG backbone by means ofan amide linkage.

In some embodiments, the hydroxylamine-terminal PEG derivatives have thestructure:RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—O—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivatives have the structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—X—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is100-1,000 and X is optionally a carbonyl group (C═O) that can be presentor absent.

In some embodiments, the semicarbazide-containing PEG derivatives havethe structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—NH—C(O)—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, an ABP comprising acarbonyl-containing amino acid is modified with a branched PEGderivative that contains a terminal hydrazine, hydroxylamine, hydrazideor semicarbazide moiety, with each chain of the branched PEG having a MWranging from 10-40 kDa and, more preferably, from 5-20 kDa.

In another embodiment of the invention, an ABP comprising anon-naturally encoded amino acid is modified with a PEG derivativehaving a branched structure. For instance, in some embodiments, thehydrazine- or hydrazide-terminal PEG derivative will have the followingstructure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000, and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the PEG derivatives containing a semicarbazidegroup will have the structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—NH—C(O)—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

In some embodiments, the PEG derivatives containing a hydroxylaminegroup will have the structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—O—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

The degree and sites at which the water soluble polymer(s) are linked tothe ABP can modulate the binding of the ABP to an antigen or receptor.

Methods and chemistry for activation of polymers as well as forconjugation of peptides are described in the literature and are known inthe art. Commonly used methods for activation of polymers include, butare not limited to, activation of functional groups with cyanogenbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin,divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc.(see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION. FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S. S. Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES,Academic Press, N.Y.; Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American ChemicalSociety, Washington, D.C. 1991).

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example, Harris, Macronol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and WO 93/15189, and for conjugation between activatedpolymers and enzymes including but not limited to Coagulation FactorVIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrying molecule(U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase(Veronese at al., App. Biochem. Biotech. 11: 141-45 (1985)). Allreferences and patents cited are incorporated by reference herein.

PEGylation (i.e., addition of any water soluble polymer) ofantigen-binding polypeptides containing a non-naturally encoded aminoacid, such as p-azido-L-phenylalanine, is carried out by any convenientmethod. For example, ABP is PEGylated with an alkyne-terminated mPEGderivative. Briefly, an excess of solid mPEG(5000)-O—CH₂—C≡CH is added,with stirring, to an aqueous solution of p-azido-L-Phe-containing ABP atroom temperature. Typically, the aqueous solution is buffered with abuffer having a pK_(a) near the pH at which the reaction is to becarried out (generally about pH 4-10). Examples of suitable buffers forPEGylation at pH 7.5, for instance, include, but are not limited to,HEPES, phosphate, borate, TRIS-HCl, EPPS, and TES. The pH iscontinuously monitored and adjusted if necessary. The reaction istypically allowed to continue for between about 1-48 hours.

The reaction products are subsequently subjected to hydrophobicinteraction chromatography to separate the PEGylated ABP variants fromfree mPEG(5000)-O—CH₂—C≡CH and any high-molecular weight complexes ofthe pegylated ABP which may form when unblocked PEG is activated at bothends of the molecule, thereby crosslinking ABP variant molecules. Theconditions during hydrophobic interaction chromatography are such thatfree mPEG(5000)-O—CH₂—C≡CH flows through the column, while anycrosslinked PEGylated ABP variant complexes elute after the desiredforms, which contain one ABP variant molecule conjugated to one or morePEG groups. Suitable conditions vary depending on the relative sizes ofthe cross-linked complexes versus the desired conjugates and are readilydetermined by those skilled in the art. The eluent containing thedesired conjugates is concentrated by ultrafiltration and desalted bydiafiltration.

If necessary, the PEGylated ABP obtained from the hydrophobicchromatography can be purified further by one or more procedures knownto those skilled in the art including, but are not limited to, affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;reverse phase HPLC; gel filtration (using, including but not limited to,SEPHADEX G-75); hydrophobic interaction chromatography; size-exclusionchromatography, metal-chelate chromatography;ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfateprecipitation; chromatofocusing; displacement chromatography;electrophoretic procedures (including but not limited to preparativeisoelectric focusing), differential solubility (including but notlimited to ammonium sulfate precipitation), or extraction. Apparentmolecular weight may be estimated by GPC by comparison to globularprotein standards (PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH(Harris & Angal, Eds.) IRL Press 1989, 293-306). The purity of theABP-PEG conjugate can be assessed by proteolytic degradation (includingbut not limited to, trypsin cleavage) followed by mass spectrometryanalysis. Pepinsky B., et al., J. Pharmcol. & Exp. Ther. 297(3):1059-66(2001).

A water soluble polymer linked to an amino acid of an ABP of theinvention can be further derivatized or substituted without limitation.

Azide-Containing Peg Derivatives

In another embodiment of the invention, an antigen-binding polypeptideis modified with a PEG derivative that contains an azide moiety thatwill react with an alkyne moiety present on the side chain of thenon-naturally encoded amino acid. In general, the PEG derivatives willhave an average molecular weight ranging from 1-100 kDa and, in someembodiments, from 10-40 kDa.

In some embodiments, the azide-terminal PEG derivative will have thestructure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—N₃where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment, the azide-terminal PEG derivative will have thestructure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—N₃where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000 (i.e., average molecular weight is between 5-40kDa).

In another embodiment of the invention, an ABP comprising aalkyne-containing amino acid is modified with a branched PEG derivativethat contains a terminal azide moiety, with each chain of the branchedPEG having a MW ranging from 10-40 kDa and, more preferably, from 5-20kDa. For instance, in some embodiments, the azide-terminal PEGderivative will have the following structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)N₃where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), in each case that can be present or absent.Alkyne-Containing Peg Derivatives

In another embodiment of the invention, an antigen-binding polypeptideis modified with a PEG derivative that contains an alkyne moiety thatwill react with an azide moiety present on the side chain of thenon-naturally encoded amino acid.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—C≡CHwhere R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment of the invention, an antigen-binding polypeptidecomprising an alkyne-containing non-naturally encoded amino acid ismodified with a PEG derivative that contains a terminal azide orterminal alkyne moiety that is linked to the PEG backbone by means of anamide linkage.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—C≡CHwhere R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000.

In another embodiment of the invention, an antigen-binding polypeptidecomprising an azide-containing amino acid is modified with a branchedPEG derivative that contains a terminal alkyne moiety, with each chainof the branched PEG having a MW ranging from 1040 kDa and, morepreferably, from 5-20 kDa. For instance, in some embodiments, thealkyne-terminal PEG derivative will have the following structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)C≡CHwhere R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), or not present.Phosphine-Containing PEG Derivatives

In another embodiment of the invention, an antigen-binding polypeptideis modified with a PEG derivative that contains an activated functionalgroup (including but not limited to, ester, carbonate) furthercomprising an aryl phosphine group that will react with an azide moietypresent on the side chain of the non-naturally encoded amino acid. Ingeneral, the PEG derivatives will have an average molecular weightranging from 1-100 kDa and, in some embodiments, from 10-40 kDa.

In some embodiments, the PEG derivative will have the structure:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

In some embodiments, the PEG derivative will have the structure:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).Other PEG Derivatives and General PEGylation Techniques

Other exemplary PEG molecules that may be linked to antigen-bindingpolypeptides, as well as PEGylation methods include those described in,e.g., U.S. Patent Publication No. 2004/0001838; 2002/0052009;2003/0162949; 2004/0013637; 2003/0228274; 2003/0220447; 2003/0158333;2003/0143596; 2003/0114647; 2003/0105275; 2003/0105224; 2003/0023023;2002/0156047; 2002/0099133; 2002/0086939; 2002/0082345; 2002/0072573;2002/0052430; 2002/0040076; 2002/0037949; 2002/0002250; 2001/0056171;2001/0044526; 2001/0027217; 2001/0021763; U.S. Pat. Nos. 6,646,110;5,824,778; 5,476,653; 5,219,564; 5,629,384; 5,736,625; 4,902,502;5,281,698; 5,122,614; 5,473,034; 5,516,673; 5,382,657; 6,552,167;6,610,281; 6,515,100; 6,461,603; 6,436,386; 6,214,966; 5,990,237;5,900,461; 5,739,208; 5,672,662; 5,446,090; 5,808,096; 5,612,460;5,324,844; 5,252,714; 6,420,339; 6,201,072; 6,451,346; 6,306,821;5,559,213; 5,612,460; 5,747,646; 5,834,594; 5,849,860; 5,980,948;6,004,573; 6,129,912; WO 97/32607, EP 229,108, EP 402,378, WO 92/16555,WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO97/03106, WO 96/21469, WO 95/13312, EP 921 131, WO 98/05363, EP 809 996,WO 96/41813, WO 96/07670, EP 605 963, EP 510 356, EP 400 472, EP 183 503and EP 154 316, which are incorporated by reference herein. Any of thePEG molecules described herein may be used in any form, including butnot limited to, single chain, branched chain, multiarm chain, singlefunctional, bi-functional, multi-functional, or any combination thereof.

Enhancing Affinity for Serum Albumin

Various molecules can also be fused to the antigen-binding polypeptidesof the invention to modulate the half-life of ABP in serum. In someembodiments, molecules are linked or fused to antigen-bindingpolypeptides of the invention to enhance affinity for endogenous serumalbumin in an animal.

For example, in some cases, a recombinant fusion of an antigen-bindingpolypeptide and an albumin binding sequence is made. Exemplary albuminbinding sequences include, but are not limited to, the albumin bindingdomain from streptococcal protein G (see. e.g., Makrides et al., J.Pharmacol. Exp. Ther. 277:534-542 (1996) and Sjolander et al., J.Immunol. Methods 201:115-123 (1997)), or albumin-binding peptides suchas those described in, e.g., Dennis, et al., J. Biol. Chem.277:35035-35043 (2002).

In other embodiments, the antigen-binding polypeptides of the presentinvention are acylated with fatty acids. In some cases, the fatty acidspromote binding to serum albumin. See, e.g., Kurtzhals, et al., Biochem.J. 312:725-731 (1995).

In other embodiments, the antigen-binding polypeptides of the inventionare fused directly with serum albumin (including but not limited to,human serum albumin). Those of skill in the art will recognize that awide variety of other molecules can also be linked to ABP in the presentinvention to modulate binding to serum albumin or other serumcomponents.

X. Glycosylation of ABP

The invention includes antigen-binding polypeptides incorporating one ormore non-naturally encoded amino acids bearing saccharide residues. Thesaccharide residues may be either natural (including but not limited to,N-acetylglucosamine) or non-natural (including but not limited to,3-fluorogalactose). The saccharides may be linked to the non-naturallyencoded amino acids either by an N- or O-linked glycosidic linkage(including but not limited to, N-acetylgalactose-L-serine) or anon-natural linkage (including but not limited to, an oxime or thecorresponding C- or S-linked glycoside).

The saccharide (including but not limited to, glycosyl) moieties can beadded to antigen-binding polypeptides either in vivo or in vitro. Insome embodiments of the invention, a nantigen-binding polypeptidecomprising a carbonyl-containing non-naturally encoded amino acid ismodified with a saccharide derivatized with an aminooxy group togenerate the corresponding glycosylated polypeptide linked via an oximelinkage. Once attached to the non-naturally encoded amino acid, thesaccharide may be further elaborated by treatment withglycosyltransferases and other enzymes to generate an oligosaccharidebound to the antigen-binding polypeptide. See, e.g., H. Liu, et al. J.Am. Chem. Soc. 125: 1702-1703 (2003).

In some embodiments of the invention, an antigen-binding polypeptidecomprising a carbonyl-containing non-naturally encoded amino acid ismodified directly with a glycan with defined structure prepared as anaminooxy derivative. One skilled in the art will recognize that otherfunctionalities, including azide, alkyne, hydrazide, hydrazine, andsemicarbazide, can be used to link the saccharide to the non-naturallyencoded amino acid.

In some embodiments of the invention, an antigen-binding polypeptidecomprising an azide or alkynyl-containing non-naturally encoded aminoacid can then be modified by, including but not limited to, a Huisgen[3+2]cycloaddition reaction with, including but not limited to, alkynylor azide derivatives, respectively. This method allows for proteins tobe modified with extremely high selectivity.

XI. ABP Dimers and Multimers

The present invention also provides for ABP combinations including butnot limited to ABP homodimers, heterodimers, homomultimers, orheteromultimers (i.e., trimers, tetramers, etc.) where an ABPpolypeptide containing one or more non-naturally encoded amino acids isbound to another ABP or variant thereof or any other polypeptide that isnon-ABP polypeptide or variant thereof, either directly to thepolypeptide backbone or via a linker. Due to its increased molecularweight compared to monomers, the ABP dimer or multimer conjugates mayexhibit new or desirable properties, including but not limited todifferent pharmacological, pharmacokinetic, pharmacodynamic, modulatedtherapeutic half-life, or modulated plasma half-life relative to themonomeric ABP. In some embodiments, the ABP dimers of the invention willmodulate the dimerization of the ABP receptor. In other embodiments, theABP dimers or multimers of the present invention will act as an ABPreceptor antagonist, agonist, or modulator.

In some embodiments, one or more of ABP present in an ABP containingdimer or multimer comprises a non-naturally encoded amino acid linked toa water soluble polymer. In some embodiments, the ABPs are linkeddirectly, including but not limited to, via an Asn-Lys amide linkage orCys-Cys disulfide linkage. In some embodiments, the linked ABPs, and/orthe linked non-ABP polypeptide, will comprise different non-naturallyencoded amino acids to facilitate dimerization, including but notlimited to, an alkyne in one non-naturally encoded amino acid of a firstABP and an azide in a second non-naturally encoded amino acid of asecond ABP will be conjugated via a Huisgen [3+2]cycloaddition.Alternatively, a first ABP, and/or the linked non-ABP polypeptidecomprising a ketone-containing non-naturally encoded amino acid can beconjugated to a second ABP polypeptide comprising ahydroxylamine-containing non-naturally encoded amino acid and thepolypeptides are reacted via formation of the corresponding oxime.

Alternatively, the two ABPs, and/or the linked non-ABP polypeptide, arelinked via a linker. Any hetero- or homo-bifunctional linker can be usedto link the two ABPs, and/or the linked non-ABP polypeptides, which canhave the same or different primary sequence. In some cases, the linkerused to tether the ABP, and/or the linked non-ABP polypeptides togethercan be a bifunctional PEG reagent. The linker may have a wide range ofmolecular weight or molecular length. Larger or smaller molecular weightlinkers may be used to provide a desired spatial relationship orconformation between the ABP and the linked entity. Linkers havinglonger or shorter molecular length may also be used to provide a desiredspace or flexibility between the ABP and the linked entity. Similarly, alinker having a particular shape or conformation may be utilized toimpart a particular shape or conformation to the ABP or the linkedentity, either before or after the ABP reaches its target. Thefunctional groups present on each end of the linker may be selected tomodulate the release of an ABP or a non-ABP polypeptide under desiredconditions. This optimization of the spatial relationship between theABP and the linked entity may provide new, modulated, or desiredproperties to the molecule.

In some embodiments, the invention provides water-soluble bifunctionallinkers that have a dumbbell structure that includes: a) an azide, analkyne, a hydrazine, a hydrazide, a hydroxylamine, or acarbonyl-containing moiety on at least a first end of a polymerbackbone; and b) at least a second functional group on a second end ofthe polymer backbone. The second functional group can be the same ordifferent as the first functional group. The second functional group, insome embodiments, is not reactive with the first functional group. Theinvention provides, in some embodiments, water-soluble compounds thatcomprise at least one arm of a branched molecular structure. Forexample, the branched molecular structure can be dendritic.

In some embodiments, the invention provides multimers comprising one ormore ABP formed by reactions with water soluble activated polymers thathave the structure:R—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—Xwherein n is from about 5 to 3,000, m is 2-10, X can be an azide, analkyne, a hydrazine, a hydrazide, an aminooxy group, a hydroxylamine, aacetyl, or carbonyl-containing moiety, and R is a capping group, afunctional group, or a leaving group that can be the same or differentas X. R can be, for example, a functional group selected from the groupconsisting of hydroxyl, protected hydroxyl, alkoxyl,N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester,N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal,aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, and ketone.XII. Measurement of ABP Activity and Affinity of ABP for the ABP Antigenor Binding Partner

ABP activity can be determined using standard in vitro or in vivoassays. For example, cells or cell lines that bind ABP (including butnot limited to, cells containing native ABP antigen or binding partneror recombinant ABP antigen or binding partner producing cells) can beused to monitor ABP binding. For a non-PEGylated or PEGylatedantigen-binding polypeptide comprising a non-natural amino acid, theaffinity of the ABP for its antigen or binding partner can be measuredby using techniques known in the art such as a BIAcore™ biosensor(Pharmacia).

Regardless of which methods are used to create the ABP's, the ABP's aresubject to assays for biological activity. Tritiated thymidine assaysmay be conducted to ascertain the degree of cell division, ifappropriate. Other biological assays, however, may be used to ascertainthe desired activity. Biological assays such as measuring the ability toinhibit an antigen's biological activity, such as an enzymatic,proliferative, or metabolic activity also provides an indication of ABPactivity. Other in vitro assays may be used to ascertain biologicalactivity. In general, the test for biological activity should provideanalysis for the desired result, such as increase or decrease inbiological activity (as compared to non-altered ABP), differentbiological activity (as compared to non-altered ABP), receptor affinityanalysis, conformational or structural changes, or serum half-lifeanalysis, as appropriate for the antigen's biological activity.

The above compilation of references for assay methodologies is notexhaustive, and those skilled in the art will recognize other assaysuseful for testing for the desired end result.

XIII. Measurement of Potency, Functional In Vivo Half-Life, andPharmacokinetic Parameters

An important aspect of the invention is the prolonged biologicalhalf-life that is obtained by construction of ABP with or withoutconjugation of the ABP to a water soluble polymer moiety. The rapiddecrease of ABP serum concentrations has made it important to evaluatebiological responses to treatment with conjugated and non-conjugated ABPand variants thereof. Preferably, the conjugated and non-conjugated ABPand variants thereof of the present invention have prolonged serumhalf-lives also after i.v. administration, making it possible to measureby, e.g. ELISA method or by a primary screening assay. Measurement of invivo biological half-life is carried out as described herein.

Pharmacokinetic parameters for an antigen-binding polypeptide comprisinga non-naturally encoded amino acid can be evaluated in normalSprague-Dawley male rats (N=5 animals per treatment group). Animals willreceive either a single dose of 25 ug/rat iv or 50 ug/rat sc, andapproximately 5-7 blood samples will be taken according to a pre-definedtime course, generally covering about 6 hours for a n antigen-bindingpolypeptide comprising a non-naturally encoded amino acid not conjugatedto a water soluble polymer and about 4 days for an antigen-bindingpolypeptide comprising a non-naturally encoded amino acid and conjugatedto a water soluble polymer. Pharmacokinetic data for ABP is well-studiedin several species and can be compared directly to the data obtained forABP comprising a non-naturally encoded amino acid.

The specific activity of ABP in accordance with this invention can bedetermined by various assays known in the art. The biological activityof the ABP muteins, or fragments thereof, obtained and purified inaccordance with this invention can be tested by methods described orreferenced herein or known to those skilled in the art.

XIV. Administration and Pharmaceutical Compositions

The polypeptides or proteins of the invention (including but not limitedto, ABP, synthetases, proteins comprising one or more unnatural aminoacid, etc.) are optionally employed for therapeutic uses, including butnot limited to, in combination with a suitable pharmaceutical carrier.Such compositions, for example, comprise a therapeutically effectiveamount of the compound, and a pharmaceutically acceptable carrier orexcipient. Such a carrier or excipient includes, but is not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, and/orcombinations thereof. The formulation is made to suit the mode ofadministration. In general, methods of administering proteins are wellknown in the art and can be applied to administration of thepolypeptides of the invention.

Therapeutic compositions comprising one or more polypeptide of theinvention are optionally tested in one or more appropriate in vitroand/or in vivo animal models of disease, to confirm efficacy, tissuemetabolism, and to estimate dosages, according to methods well known inthe art. In particular, dosages can be initially determined by activity,stability or other suitable measures of unnatural herein to naturalamino acid homologues (including but not limited to, comparison of anABP modified to include one or more unnatural amino acids to a naturalamino acid ABP), i.e., in a relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The unnaturalamino acid polypeptides of the invention are administered in anysuitable manner, optionally with one or more pharmaceutically acceptablecarriers. Suitable methods of administering such polypeptides in thecontext of the present invention to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

Polypeptide compositions can be administered by a number of routesincluding, but not limited to oral, intravenous, intraperitoneal,intramuscular, transdermal, subcutaneous, topical, sublingual, or rectalmeans. Compositions comprising non-natural amino acid polypeptides,modified or unmodified, can also be administered via liposomes. Suchadministration routes and appropriate formulations are generally knownto those of skill in the art.

The ABP comprising a non-natural amino acid, alone or in combinationwith other suitable components, can also be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of packaged ABP can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for natural amino acid homologue therapeutics (includingbut not limited to, those typically used for EPO, GH, ABP, G-CSF,GM-CSF, IFNs, interleukins, antibodies, and/or any otherpharmaceutically delivered protein), along with formulations in currentuse, provide preferred routes of administration and formulation for thepolypeptides of the invention.

The dose administered to a patient, in the context of the presentinvention, is sufficient to have a beneficial therapeutic response inthe patient over time, or, including but not limited to, to inhibitinfection by a pathogen, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularvector, or formulation, and the activity, stability or serum half-lifeof the unnatural amino acid polypeptide employed and the condition ofthe patient, as well as the body weight or surface area of the patientto be treated. The size of the dose is also determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, formulation, or the like in aparticular patient.

In determining the effective amount of the vector or formulation to beadministered in the treatment or prophylaxis of disease (including butnot limited to, cancers, inherited diseases, diabetes, AIDS, or thelike), the physician evaluates circulating plasma levels, formulationtoxicities, progression of the disease, and/or where relevant, theproduction of anti-unnatural amino acid polypeptide antibodies.

The dose administered, for example, to a 70 kilogram patient, istypically in the range equivalent to dosages of currently-usedtherapeutic proteins, adjusted for the altered activity or serumhalf-life of the relevant composition. The vectors of this invention cansupplement treatment conditions by any known conventional therapy,including antibody administration, vaccine administration,administration of cytotoxic agents, natural amino acid polypeptides,nucleic acids, nucleotide analogues, biologic response modifiers, andthe like.

For administration, formulations of the present invention areadministered at a rate determined by the LD-50 or ED-50 of the relevantformulation, and/or observation of any side-effects of the unnaturalamino acids at various concentrations, including but not limited to, asapplied to the mass and overall health of the patient. Administrationcan be accomplished via single or divided doses.

If a patient undergoing infusion of a formulation develops fevers,chills, or muscle aches, he/she receives the appropriate dose ofaspirin, ibuprofen, acetaminophen or other pain/fever controlling drug.Patients who experience reactions to the infusion such as fever, muscleaches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, including but notlimited to, diphenhydramine. Meperidine is used for more severe chillsand muscle aches that do not quickly respond to antipyretics andantihistamines. Cell infusion is slowed or discontinued depending uponthe severity of the reaction.

Human antigen-binding polypeptides of the invention can be administereddirectly to a mammalian subject. Administration is by any of the routesnormally used for introducing ABP to a subject. The ABP compositionsaccording to embodiments of the present invention include those suitablefor oral, rectal, topical, inhalation (including but not limited to, viaan aerosol), buccal (including but not limited to, sub-lingual),vaginal, parenteral (including but not limited to, subcutaneous,intramuscular, intradermal, intraarticular, intrapleural,intraperitoneal, inracerebral, intraarterial, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated. Administration can be either local or systemic. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials. ABP of the invention canbe prepared in a mixture in a unit dosage injectable form (including butnot limited to, solution, suspension, or emulsion) with apharmaceutically acceptable carrier. ABP of the invention can also beadministered by continuous infusion (using, including but not limitedto, minipumps such as osmotic pumps), single bolus or slow-release depotformulations.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

The pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of pharmaceutical compositions (including optionalpharmaceutically acceptable carriers, excipients, or stabilizers) of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed. 1985)).

Suitable carriers include buffers containing phosphate, borate, HEPES,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates, including glucose, mannose, ordextrins; chelating agents such as EDTA; divalent metal ions such aszinc, cobalt, or copper; sugar alcohols such as mannitol or sorbitol;salt-forming counter ions such as sodium; and/or nonionic surfactantssuch as Tween™, Pluronics™, or PEG.

ABP's of the invention, including those linked to water soluble polymerssuch as PEG can also be administered by or as part of sustained-releasesystems. Sustained-release compositions include, including but notlimited to, semi-permeable polymer matrices in the form of shapedarticles, including but not limited to, films, or microcapsules.Sustained-release matrices include from biocompatible materials such aspoly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res., 15: 167-277 (1981); Langer, Chem. Tech., 12: 98-105 (1982),ethylene vinyl acetate (Langer et al., supra) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988), polylactides (polylacticacid) (U.S. Pat. No. 3,773,919; EP 58,481), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (copolymers of lactic acid andglycolic acid) polyanhydrides, copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers, 22, 547-556(1983), poly(ortho)esters, polypeptides, hyaluronic acid, collagen,chondroitin sulfate, carboxylic acids, fatty acids, phospholipids,polysaccharides, nucleic acids, polyamino acids, amino acids such asphenylalanine, tyrosine, isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone and silicone. Sustained-releasecompositions also include a liposomally entrapped compound. Liposomescontaining the compound are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034(1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324. All references and patents cited are incorporated byreference herein.

Liposomally entrapped ABP can be prepared by methods described in, e.g.,DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Composition and size of liposomes are wellknown or able to be readily determined empirically by one skilled in theart. Some examples of liposomes as described in, e.g., Park J W, et al.,Proc. Natl. Acad. Sci. USA 92:1327-1331 (1995); Lasic D andPapahadjopoulos D (eds): MEDICAL APPLICATIONS OF LIPOSOMES (1998);Drummond D C, et al., Liposomal drug delivery systems for cancertherapy, in Teicher B (ed): CANCER DRUG DISCOVERY AND DEVELOPMENT(2002); Park J W, et al., Clin. Cancer Res. 8:1172-1181 (2002); NielsenU B, et al., Biochim. Biophys. Acta 1591(1-3):109-118 (2002); Mamot C,et al., Cancer Res. 63: 3154-3161 (2003). All references and patentscited are incorporated by reference herein.

The dose administered to a patient in the context of the presentinvention should be sufficient to cause a beneficial response in thesubject over time. Generally, the total pharmaceutically effectiveamount of the ABP of the present invention administered parenterally perdose is in the range of about 0.01 μg/kg/day to about 100 μg/kg, orabout 0.05 mg/kg to about 1 mg/kg, of patient body weight, although thisis subject to therapeutic discretion. The frequency of dosing is alsosubject to therapeutic discretion, and may be more frequent or lessfrequent than the commercially available ABP products approved for usein humans. Generally, a PEGylated antigen-binding polypeptide of theinvention can be administered by any of the routes of administrationdescribed above.

XV. Therapeutic Uses of Antigen-Binding Polypeptides of the Invention

The ABP polypeptides of the invention are useful for treating a widerange of disorders. The pharmaceutical compositions containing the ABPmay be formulated at a strength effective for administration by variousmeans to a human patient experiencing disorders that may be affected byABP agonists or antagonists, such as but not limited to,anti-proliferatives, anti-inflammatory, or anti-virals are used, eitheralone or as part of a condition or disease. Average quantities of ABPmay vary and in particular should be based upon the recommendations andprescription of a qualified physician. The exact amount of ABP is amatter of preference subject to such factors as the exact type ofcondition being treated, the condition of the patient being treated, aswell as the other ingredients in the composition. The invention alsoprovides for administration of a therapeutically effective amount ofanother active agent such as an anti-cancer chemotherapeutic agent. Theamount to be given may be readily determined by one skilled in the artbased upon therapy with ABP.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

This example describes one of the many potential sets of criteria forthe selection of preferred sites of incorporation of non-naturallyencoded amino acids into ABP.

This example demonstrates how preferred sites within the antigen-bindingpolypeptide were selected for introduction of a non-naturally encodedamino acid. The three dimensional structure composed of two molecules ofABP, or the secondary, tertiary, or quaternary structure of ABP was usedto determine preferred positions into which one or more non-naturallyencoded amino acids could be introduced.

The following criteria were used to evaluate each position of ABP forthe introduction of a non-naturally encoded amino acid: the residue (a)should not interfere with binding of either ABP based on structuralanalysis of three dimensional structures, or the secondary, tertiary, orquaternary structure of ABP, b) should not be affected by alanine orhomolog scanning mutagenesis (c) should be surface exposed and exhibitminimal van der Waals or hydrogen bonding interactions with surroundingresidues, (d) may be on one or more of the exposed faces of ABP, (e) maybe a site or sites of ABP that are juxtaposed to a second ABP, or othermolecule or fragment thereof, (f) should be either deleted or variablein ABP variants, (g) would result in conservative changes uponsubstitution with a non-naturally encoded amino acid, (h) may modulatethe conformation of the ABP itself or a dimer or multimer comprising oneor more ABP, by altering the flexibility or rigidity of the completestructure as desired, (i) could be found in either highly flexibleregions or structurally rigid regions and (j) are found incomplementarity determining regions (CDR) or not. In addition, furthercalculations were performed on the ABP molecule, utilizing the Cxprogram (Pintar et al. Bioinformatics, 18, pp 980) to evaluate theextent of protrusion for each protein atom. As a result, in someembodiments, the non-naturally encoded amino acid is substituted at, butnot limited to, one or more positions of ABP.

Example 2

This example details cloning and expression of ABP including anon-naturally encoded amino acid in E. coli.

An introduced translation system that comprises an orthogonal tRNA(O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O—RS) is used toexpress ABP containing a non-naturally encoded amino acid. The O—RSpreferentially aminoacylates the O-tRNA with a non-naturally encodedamino acid. In turn the translation system inserts the non-naturallyencoded amino acid into ABP, in response to an encoded selector codon.TABLE 2 O-RS and O-tRNA sequences. SEQ ID NO: 1 M. jannaschiimtRNA_(CUA) ^(Tyr) tRNA SEQ ID NO: 2 HLAD03; an optimized ambersupressor tRNA tRNA SEQ ID NO: 3 HL325A; an optimized AGGA frameshiftsupressor tRNA tRNA SEQ ID NO: 4 Aminoacyl tRNA synthetase for theincorporation of p-azido-L-phenylalanine RS p-Az-PheRS(6) SEQ ID NO: 5Aminoacyl tRNA synthetase for the incorporation ofp-benzoyl-L-phenylalanine RS p-BpaRS(1) SEQ ID NO: 6 Aminoacyl tRNAsynthetase for the incorporation of propargyl-phenylalanine RSPropargyl-PheRS SEQ ID NO: 7 Aminoacyl tRNA synthetase for theincorporation of propargyl-phenylalanine RS Propargyl-PheRS SEQ ID NO: 8Aminoacyl tRNA synthetase for the incorporation ofpropargyl-phenylalanine RS Propargyl-PheRS SEQ ID NO: 9 Aminoacyl tRNAsynthetase for the incorporation of p-azido-phenylalanine RSp-Az-PheRS(1) SEQ ID NO: 10 Aminoacyl tRNA synthetase for theincorporation of p-azido-phenylalanine RS p-Az-PheRS(3) SEQ ID NO: 11Aminoacyl tRNA synthetase for the incorporation of p-azido-phenylalanineRS p-Az-PheRS(4) SEQ ID NO: 12 Aminoacyl tRNA synthetase for theincorporation of p-azido-phenylalanine RS p-Az-PheRS(2) SEQ ID NO: 13Aminoacyl tRNA synthetase for the incorporation ofp-acetyl-phenylalanine (LW1) RS SEQ ID NO: 14 Aminoacyl tRNA synthetasefor the incorporation of p-acetyl-phenylalanine (LW5) RS SEQ ID NO: 15Aminoacyl tRNA synthetase for the incorporation ofp-acetyl-phenylalanine (LW6) RS SEQ ID NO: 16 Aminoacyl tRNA synthetasefor the incorporation of p-azido-phenylalanine (AzPheRS-5) RS SEQ ID NO:17 Aminoacyl tRNA synthetase for the incorporation ofp-azido-phenylalanine (AzPheRS-6) RS

The transformation of E. coli with plasmids containing the modified ABPgene and the orthogonal aminoacyl tRNA synthetase/tRNA pair (specificfor the desired non-naturally encoded amino acid) allows thesite-specific incorporation of non-naturally encoded amino acid into theABP. The transformed E. coli, grown at 37° C. in media containingbetween 0.01-100 mM of the particular non-naturally encoded amino acid,expresses modified ABP with high fidelity and efficiency. The His-taggedABP containing a non-naturally encoded amino acid is produced by the E.coli host cells as inclusion bodies or aggregates. The aggregates aresolubilized and affinity purified under denaturing conditions in 6Mguanidine HCl. Refolding is performed by dialysis at 4° C. overnight in50 mM TRIS-HCl, pH8.0, 40 μM CuSO₄, and 2% (w/v) Sarkosyl. The materialis then dialyzed against 20 mM TRIS-HCl, pH 8.0, 100 mM NaCl, 2 mMCaCl₂, followed by removal of the His-tag. See Boissel et al., (1993)268:15983-93. Methods for purification of ABP are well known in the artand are confirmed by SDS-PAGE, Western Blot analyses, orelectrospray-ionization ion trap mass spectrometry and the like.

Expression Constructs

Periplasmic scFv-108: The variable regions (VL and VH) of theEGFR-specific monoclonal antibody mAb108 (U.S. Pat. No. 6,217,866 whichis incorporated by reference herein) were cloned as a scFv fragment with(GGGGS)₄ linker sequence downstream of a yeast BGL2 (C7) periplasmicleader sequence (Humphreys, D P et al. Protein Expr Purif. 2000November; 20(2):252-64). An epitope sequence recognized by the c-mycantibody as well as a 6×-His tag were cloned downstream of the VLdomain. The wild type scFv-108 construct, as well as variants containingthe amber stop codon (TAG) in the VL domain (see FIG. 2, Panel A) werecloned into an E. coli expression vector under the control of aninducible promoter. This plasmid also constitutively expressed an ambersuppressor tyrosyl tRNA^(Tyr/CUA) from Methanococcus jannaschii (MjtRNA^(Tyr/CUA)). Locations of the amber stop codons are indicated. Theconstruct made is shown as SEQ ID NO: 18 (nucleotide sequence) and SEQID NO: 19 (translated protein sequence). The construct is described inTables 3 and 4. TABLE 3 Sequence Position (SEQ ID NO: 18) C7 leader15-83 VH  84-446 GlySer linker 447-506 VL 507-827 Myc 843-887 His888-905 TAA 906-908

TABLE 4 Sequence Position (SEQ ID NO: 19) C7 leader  1-23 VH  24-144GlySer linker 145-164 VL 165-276 Myc 277-291 His 292-297

Cytoplasmic scFv-108: VH-linker VL (VH-GlySer-VL) sequences containingan N-terminal MetGly-sequence and a 6×-His sequence were cloned into anexpression vector under control of the T7 promoter (see FIG. 2, PanelB). Location of the amber stop codons and PEGylated residues areindicated. The construct made is shown as SEQ ID NO: (nucleotidesequence) and SEQ ID NO: 21 (translated protein sequence). The constructis described in Table 5. TABLE 5 Sequence Position (SEQ ID NO: 20) His 3-26 VH  27-389 GlySer linker 390-449 VL 450-767

Fab-108: The VL and VH sequences of mAb108 were cloned into pFT3, aplasmid encoding the g3 (VL) and STII (VH) periplasmic leader sequences,as well as the human κ constant and CH1 domains. The C-terminus of theCH1 domain contained a 6-His tag to facilitate purification. Ambermutations were introduced into the CH1 domain, and the entirebicistronic cassette was cloned into the expression plasmid thatconstitutively expressed an amber suppressor tyrosyl tRNA^(Tyr/CUA) fromMethanococcus jannaschii (Mj tRNA^(Tyr/CUA)) (see FIG. 2, Panel C). Thetwo Shine Delgarno sequences (SD) driving translation of the VL and VHdomains of the Fab fragment are shown. The construct made is shown asSEQ ID NO: 22 (nucleotide sequence) and SEQ ID NO: 23 and 24 (translatedprotein sequence of VL Kappa chain of Fab 108; VH-CH1 chain of Fab108).The construct is described in Table 6. TABLE 6 Sequence Position (SEQ IDNO: 22) g3 leader 15-68 VL  81-416 K chain 432-755 STII leader 788-856VH  875-1237 CH1 domain 1247-1543 His 1544-1561

Periplasmic scFv-4D5: The variable regions (VL and VH) of theHER2-specific monoclonal antibody mAb-4D5 (Carter, P., et. al.,Biotechnology (N Y). 1992 February; 10(2):163-7) were cloned as scFvfragments downstream of a yeast BGL2 (C7) periplasmic leader sequence. A6×-His tag was cloned either at the C-terminus of the VL sequence(scFv-4D5-His; FIG. 2, Panel D), or at the N-terminus of the VH domain(His-scFv-4D5; FIG. 2, Panel E). The wild type scFv-4D5 constructs, aswell as a variant containing the amber stop codon (TAG) in the GlySerlinker domain were cloned into the E. coli expression vector thatconstitutively expressed an amber suppressor tyrosyl tRNA^(Tyr/CUA) fromMethanococcus jannaschii (Mj tRNA^(Tyr/CUA)). The 6×-His C terminalconstruct made is shown as SEQ ID NO: 25 (nucleotide sequence) and SEQID NO: 26 (translated protein sequence). The 6×-His N terminal constructmade is shown as SEQ ID NO: 27 (nucleotide sequence) and SEQ ID NO: 28(translated protein sequence). The 6×-His C terminal construct isdescribed in Table 7, and the 6×-His N terminal construct is describedin Table 8. TABLE 7 Sequence Position (SEQ ID NO: 25) C7 leader 15-83 VH 84-443 GlySer linker 444-503 VL 504-824 His 825-848

TABLE 8 Sequence Position (SEQ ID NO: 27) C7 leader 15-83 His  84-107 VH108-470 GlySer linker 471-530 VL 531-854

Fab-4D5: The VL and VH sequences of mAb 4D5 were subcloned into pFT3, aplasmid encoding the g3 and STII periplasmic leader sequences, as wellas the human κ constant and CH1 domains, and then were cloned into theexpression plasmid that constitutively expressed an amber suppressortyrosyl tRNA^(Tyr/CUA) from Methanococcus jannaschii (MjtRNA^(Tyr/CUA)). FIG. 2, Panel F shows the cistron used for expressionof Fab-4D5. An amber mutation was introduced into the CH1 domain of Fab4D5 at lysine 139. This lysine corresponds to K142 in Fab 108. A Fab-4D5construct containing an extra cysteine residue (THTCAA) at theC-terminus of the CH1 domain was made by overlapping PCR (Fab-4D5-cys).The construct made is shown as SEQ ID NO: 29 (nucleotide sequence) andSEQ ID NO: 30 and 31 (translated protein sequence of VL Kappa chain ofFab 4D5; VH-CH1 chain of Fab 4D5). The construct is described in Table9. TABLE 9 Sequence Position g3 leader  1-54 VL  67-386 K chain 403-726STII leader 759-827 VH  846-1205 CH1 domain 1215-1511 His 1512-1529Expression/Suppression

Suppression with para-acetyl-phenylalanine (pAcF): Suppression of theamber mutations in E. coli was achieved using standard protocols knownin the art. Briefly, for the periplasmic suppression of antibodyfragments in E. coli (scFv and Fab), the expression vector construct wastransformed into E. coli host cells with a plasmid encoding theorthogonal tyrosyl-tRNA-synthetase from M. jannaschii (MjTyrRS).Overnight bacterial cultures were diluted 1:100 into shake flaskscontaining either LB media (Luria-Bertani) or Superbroth, and grown at37° C. to an OD of approximately 0.8. Fab and scFv expression wasinduced while suppression of the amber codon was achieved by theaddition of para-acetyl-phenylalanine (pAcF) to a final concentration of4 mM. Cultures were incubated at 25° C. overnight. Expression of wildtype (lacking amber codon) scFv and Fab fragments (includingFab-4D5-cys) was performed under identical conditions.Expression/suppression of cytoplasmic scFv fragments (FIG. 2, Panel B)was achieved in a similar manner.

Suppression with aa9.2: Suppression of amber mutations with a derivativeof pAcF (aa 9.2) was achieved in a similar manner as pAcF, except thatthe orthogonal tyrosyl-tRNA-synthetase from M. jannaschii (MjTyrRS) usedwas specific for this amino acid. Suppression was achieved by theaddition of aa9.2 (4 mM) at the time of induction.

Protein Extraction and Purification

Cells were harvested by centrifugation and resuspended in periplasmicrelease buffer (50 mM NaPO₄, 20% sucrose, 1 mM EDTA, pH 8.0)supplemented with 100 ug/ml of lysozyme and incubated on ice for 30minutes. After centrifugation, antibody fragments in the supernatantwere immobilized on ProBind beads (Invitrogen; Carlsbad, Calif.) byvirtue of their His tag, the beads washed extensively with bindingbuffer and then the bound fragments eluted from the beads with 0.5 Mimidazole. Purified fragments were dialyzed in storage buffer (50 mMHEPES, 150 mM NaCl, 10% glycerol, 5% sucrose, pH 7.8). For small scaleanalysis of scFv fragments expressed in the cytoplasm, E. coli from 15ml of culture were collected by centrifugation and re-suspended in 1 mlof lysis buffer (B-PER, Pierce Biotechnology; Rockford, Ill.)supplemented with 10 ug/ml of DNase. The mixture was incubated at 37° C.for 30 minutes, diluted to 1× in Protein Loading buffer (Invitrogen;Carlsbad, Calif.) and analyzed by SDS-PAGE.

FIG. 3, Panel A shows the suppression of amber mutations in the secondserine of the GlySer linker (S131Am), and purification of thecorresponding pAcF-containing scFv is shown (FIG. 3, Panel B). TheWestern blot analysis shown as FIG. 3, Panel A demonstrates that pAcF isrequired to suppress the amber stop mutation when the cells are growneither in LB or Superbroth media. The presence of pAcF does not affectexpression of a scFv lacking the TAG stop codon (WT scFv-108). FIG. 3,Panel B shows the purification of pAcF-scFv 108-(S131) by immobilizedmetal affinity chromatography (IMAC). Estimated yield of thepAcF-containing scFv was 1.5 mg/L. Position of the scFv fragment isindicated by the arrowhead. The Coomassie gel was loaded as follows:lane 1—scFv control (1.7 ug); lane 2—IMAC pre-bind (20 ul/70 ml); lane3—IMAC void (20 ul/70 ml); lane 4—IMAC elution (5 ul/1.3 ml); lane5—NAP10 buffer exchange (10 ul/1.5 ml); lane 6—IMAC beads post-elution;lane 7—scFv control (3.4 ug).

Suppression of an amber mutation in the VL chain (L156) duringcytoplasmic expression of a scFv is shown in FIG. 4. Yields were >100mg/L of E. coli culture, and suppression of the stop codon wasabsolutely dependent on the presence of pAcF. Full length scFv isindicated by the arrowhead. Products truncated at the amber codon areindicated by a filled circle.

PEGylation/Dimerization of Antibody Fragments (1)

PEGylation: Approximately 1 mg of pAcF-scFv-108 protein was concentratedin reaction buffer (100 mM NaOAc, 150 mM NaCl, 1 mM EDTA, pH 4.0) to afinal volume of 50 ul. The reaction mixture was incubated at 28° C. for32 hours with a 100-fold molar excess of mono-functional (hydroxylamine)5K PEG (equilibrated in reaction buffer) in a final volume of 100 ul.PEGylated material was evaluated following gel electrophoresis and useddirectly in cell binding assays.

Dimerization: A similar procedure was used to dimerize pAcF-containingscFv-108 fragments. Briefly, the starting pAcF containing scFv fragmentswere concentrated to a concentration of >5 ug/ul in reaction buffer andthen incubated with a bi-functional hydroxylamine-conjugated PEG linker(364 Da). Unreactive PEG was removed by dialysis, and a fresh aliquot ofpAcF-scFv fragment (1 molar protein:protein equivalence) was added tothe mixture. The mixture was then incubated at 28° C. for another 32hours. The dimer was loaded onto a cation ion exchange column (SP-5PW)equilibrated with 20 mM sodium acetate (pH 4.0) and eluted over a NaClgradient (0-0.4M).

PEGylation and dimerization of pAcF-scFv-108 fragments is shown in FIG.5. FIG. 5, Panel A shows PEGylation (5K) of pAcF-scFv-108-(L156) andpAcF-scFv-108-(S136) and dimerization of pAcF-scFv-108-(S136). The gelwas loaded as follows: lane 1—pAcF-scFv-108-(L156) (5K PEG); lane2—pAcF-scFv-108-(S136) (5K PEG); lane 3—dimerization ofpAcF-scFv-108-(S136) (364 da PEG) linker; lane 4—dimerization ofpAcF-scFv-108-(S136); linker was not removed following the firstPEGylation reaction. Position of the mono-PEGylated scFv fragments andthe scFv-108-(S136) dimer are indicated by the single and doublearrowheads, respectively. The absence of dimerization in lane 4demonstrates that scFv were not coupled through inter-moleculardisulfide bond formation. FIG. 5, Panel C shows that the conjugation ofPEG to scFv fragments is absolutely dependent on the presence of pAcF.No PEGylation of WT scFv fragments was observed. The gel was loaded asfollows: lane 1—WT scFv 108 control; lane 2—scFv WT, in reaction buffer,no PEG, 16 hours; lane 3—scFv WT+5K PEG, in reaction buffer, 16 hours.

FIG. 6 shows SDS PAGE analysis of fractions taken during cation exchangechromatography of scFv homodimers. pAcF-scFv108-(S131) fragments werehomodimerized using a bifunctional hydroxylamine 364 dalton PEG linker.Fractions were taken at different points in the NaCl gradient (0-0.4 M)during cation exchange chromatography. The gel was loaded as follows:lane 1—marker, lane 2—pAcF-scFv108(S131)-X-pAcF-scFv108(S131) Fraction#1, lane 3—pAcF-scFv108(S131)-X-pAcF-scFv108(S131) Fraction #2, lane4—pAcF-scFv108(S131)-X-pAcF-scFv108(S131) Fraction #3.

FIG. 8, Panel A shows SDS PAGE analysis of pAcF and pAcF-PEGylated Fabfragments. Fab-108 fragments modified at K142, T204, and K219 are shown,and the efficiency of PEGylation is site specific. PEGylation ofpAcF-containing Fab fragments was performed using hydroxylamineconjugated 5K PEG.

FIG. 10, Panel A and B show suppression of an amber mutation in thesecond serine of the GlySer linker of the C-terminal (pAcF-scFv-4D5-His(S133); FIG. 10, Panel A) or N-terminal (pAcF-His-scFv-4D5(S139); FIG.10, Panel B) scFv-4D5 fragments. Expression of the scFv proteins wasinduced by the addition of 0.02% arabinose for either 5 hours orovernight (16 hours). Suppression of the amber mutation was achieved bythe concomitant addition of aa9.2 (4 mM). The suppressed product isindicated by the arrowhead, and the truncated protein by the filled-incircle. Suppression yields of greater than 50% were achieved (1.5 mg/L).The control lane loaded with scFv 108, which runs slightly higher thanscFv-4D5, is indicated (C).

Fab fragments pAcF-Fab-4D5-(K139) and Fab-4D5-cys were expressed andpurified in the same manner. FIG. 11, Panel A shows samples resolved bySDS-PAGE under both reducing and non-reducing conditions. Fab fragmentyields were as follows: pAcF-Fab-4D5-(K139), 0.37 mg/L/OD (finalOD₆₀₀=3.14) and Fab-4D5-cys 0.23 mg/L/OD (final OD₆₀₀=3.26). FIG. 11,Panel B shows a Western blot of samples (5 ul) shown in FIG. 11, Panel Ausing an anti-His antibody. The samples were run under non-reducingconditions. Multimeric VH-CH1 complexes from the Fab-4D5-cys constructare indicated with arrows. No multimeric complexes were seen withpAcF-Fab-4D5-(K139).

PEGylation/Dimerization of Antibody Fragments (2)

PEGylation: Approximately 1 mg of pAcF-scFv-108 protein was concentratedin native reaction buffer (20 mM NaOAc, 150 mM NaCl, 1 mM EDTA, pH 4.0)and denaturing reaction buffer (20 mM NaOAc, 150 mM NaCl, 1 mM EDTA, 8 MUrea, pH 4.0) to a final volume of 50 ul. The reaction mixture wasincubated at 28° C. for 32 hours with a 100-fold molar excess ofmono-functional (hydroxylamine) 5K PEG (equilibrated in correspondingreaction buffers) in a final volume of 100 ul. After 16 hours, thereaction mixture was evaluated by SDS-PAGE and used directly in cellbinding assays.

Conjugation of a polypeptide with a linker, polymer, biologically activemolecule, or other molecule under denaturing conditions may have one ormore advantages. Such advantages include, but are not limited to, easierconjugation due to the improved accessibility of the reactive group,easier refolding of the conjugated polypeptide compared tonon-conjugated polypeptide, and the ability to use polypeptide at ahigher concentration for conjugation than the polypeptide concentrationusable under non-denaturing conditions. Denaturing conditions may bedesired, for example, if the polypeptide is unstable and cannot behighly concentrated for the conjugation reaction. However, conjugationof polypeptides under denaturing conditions may result in undesirableand/or unintended sites of conjugation in polypeptides with one or morecysteines, lysines, or other amino acids upon reaction with standardcysteine-based conjugation chemistries such as maleimide chemistries, orlysine-based chemistries such as maleimide chemistries. Such undesirableand/or unintended sites of conjugation may have an impact on theactivity of the conjugated polypeptide. On the other hand, polypeptidessuch as ABP comprising one or more non-naturally encoded amino acids maybe conjugated in a site-specific manner under denaturing conditionssince the reactive groups involved in the conjugation reaction are partof a non-naturally encoded amino acid. Thus, any advantages obtainedfrom conjugation under denaturing conditions may be exploited with theuse of polypeptides comprising one or more non-naturally encoded aminoacids.

FIG. 5B shows SDS-PAGE analysis of PEGylated pAcF-scFv-(S136) andcontrol samples. The gel was loaded as follows: lane 1—pAcF-scFv-(S136);lane 2—pAcF-scFv-(S136), incubated at 28° C. for 16 hours; lane3—pAcF-scFv-(S136), 5 K PEG, incubated at 28° C. for 16 hours, nativecondition; lane 4—pAcF-scFv-(S136), 5 K PEG, incubated at 28° C. for 16hours, denaturing condition. Arrows indicate scFv and PEGylated scFv.FIG. 5C shows that the conjugation of PEG to scFv fragments isabsolutely dependent on the presence of pAcF. No PEGylation of WT scFvfragments was observed. The gel was loaded as follows: lane 1—WT scFv108 control; lane 2—scFv WT, in reaction buffer, no PEG, 16 hours; lane3—scFv WT+5K PEG, in reaction buffer, 16 hours.

Sequential dimerization: Briefly, the starting pAcF containing scFvfragments were concentrated to a concentration of 10 mg/ml in reactionbuffer and then incubated with 100 fold excess of a bi-functionalhydroxylamine-conjugated PEG linker (364 Da). The reaction mixture wasincubated for 16 hours at 28° C. Unreactive PEG was removed by dialysis,and a fresh aliquot of pAcF-scFv fragment (1 molar protein:proteinequivalence) was added to the mixture. The mixture was then incubated at28° C. for another 32 hours. See FIG. 13. The dimer was loaded onto acation ion exchange column (SP-5PW, 20 micron) equilibrated with 20 mMsodium acetate (pH 4.0) and eluted over a NaCl gradient (0-0.4M).

FIG. 14, Panel A and B show non-reducing and reducing SDS PAGE analysisof dimerization samples. The gels were loaded as follows: lane 1—finalscFv dimerization reaction mixture with 364 Da PEG bifunctional linker;lane 2—final scFv dimerization reaction control without 364 Da PEGbifunctional linker; lane 3—scFv. Arrows indicate scFv dimers and scFvmonomers. scFv dimers were synthesized only in the presence of thebifunctional linker. The absence of dimerization in lane 2 demonstratesthat the scFv were not coupled through inter-molecular disulfide bondformation. Since no difference was observed between samples analyzed onreducing and non-reducing SDS PAGE gels, the presence of thebifunctional linker did not facilitate inter-molecular disulfide bondformation.

Dimer purification: The dimerization reaction mixture was purified usingstrong cation exchange column (SP-5PW, 20 micron). Buffer A: 20 mMNaOAc, pH 4.0; buffer B: 20 mM NaOAc, 1 M NaCl, pH 4.0. scFv dimereluted at 40% B. SDS PAGE analysis (FIG. 15) of the purified dimershowed that, after one column purification, the purity of the dimer wasapproximately 90%.

An example of a hetero-bifunctional ABP of the present invention isshown in FIG. 9. Based on the known crystal structure determined for twodifferent antibody molecules (for example Herceptin and Omnitarg) thatbind to different epitopes of the same antigen (for example ErbB2),specific amino acid positions are identified such that they fit within acertain desired selection criteria. Desired selection criteria for aminoacid position in this example include the relative proximity of one ormore specific amino acid positions on each molecule. Such amino acidpositions may be desired to form the hetero-bifunctional molecule shownin FIG. 9 using a linker molecule. Specific amino acid positions on eachmolecule that fit the criteria are shown in Table 10 below, as is alinker molecule that may be used to form a hetero-bifunctional ABP.Those of ordinary skill in the art will recognize that the list is by nomeans exhaustive and is merely illustrative, and that all amino acidpositions that fit a certain desired selection criteria are contemplatedto be suitable for use in the present invention. A non-natural aminoacid of the present invention may be substituted at one or more of thesepositions in each molecule to provide the chemical functional groupsutilized for linker attachment. A wide variety of other selectioncriteria may also be utilized to identify amino acid positions to fitthe desired criteria, including but not limited to, proximity betweenthe same or different molecules, conformation change modulation,distance modulation between ABP's or molecules linked to an ABP, linkerlength or shape, surface exposure, modulation of ligand bindingcharacteristics, modulation of receptor dimerization, etc. TABLE 10Potential Linkage Sites (Heavy chain of Herceptin-Light chain ofPertuzumab) distances (Å) Asn211-Asp28 15.4 Å Asn211-Gly68 15.8 ÅAsn211-Ser30 15.8 Å Asn211-Ser67 17.8 Å Lys213-Asp28 18.0 Å Asn211-Thr6918.1 Å Lys213-Gly68 18.2 Å Lys208-Ser30 19.2 Å Lys213-Thr69 20.1 ÅLys208-Ser67 20.6 Å Lys213-Asp70 21.8 Å Thr123-Tyr92 24.9 Å Ser122-Tyr9226.2 Å Gln13-Tyr92 27.9 Å

Based on the interaction of HIV-1 neutralizing human Fab 4E10 with HIVgp41 env protein, specific amino acid positions are identified such thatthey fit within a certain desired selection criteria. Desired selectioncriteria for amino acid position in this example include residues thatwould be used for conjugation of T-20 peptide to the Fab such that thebinding of T-20 to gp41 occurs without a negative effect to the bindingand recognition of the complementarity determining regions (CDR) of 4E10to gp41. T-20, also known as DP-178, inhibits entry of HIV into cells byacting as a viral fusion inhibitor. FIG. 12 shows HIV neutralizing humanFab 4E10 with a mimic g41 peptide. Potential residues for attachment ofT-20 peptide are shown. Potential residues for incorporation ofnon-naturally encoded amino acids include, but are not limited to,Gln64—heavy chain of the Fab; Glu1—light chain of the Fab, andGln27—light chain of the Fab. Those of ordinary skill in the art willrecognize that the list is by no means exhaustive and is merelyillustrative, and that all amino acid positions that fit a certaindesired selection criteria are contemplated to be suitable for use inthe present invention. A wide variety of other selection criteria mayalso be utilized to identify amino acid positions to fit the desiredcriteria, including but not limited to, proximity between the same ordifferent molecules, conformation change modulation, distance modulationbetween ABP's or molecules linked to an ABP, inclusion of a linker,linker length or shape, surface exposure, modulation of ligand bindingcharacteristics, modulation of receptor dimerization, etc.

Example 3

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of anantigen-binding polypeptide that incorporates a ketone-containingnon-naturally encoded amino acid that is subsequently reacted with anaminooxy-containing PEG of approximately 5,000 MW. Each of the residuesidentified according to the criteria of Example 1 is separatelysubstituted with a non-naturally encoded amino acid having the followingstructure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into ABP are SEQ ID NO: 1 (muttRNA, M. jannaschiimtRNA_(CUA) ^(Tyr)), and 13, 14 or 15 (TyrRS LW1, 5, or 6) described inExample 2 above.

Once modified, the ABP variant comprising the carbonyl-containing aminoacid is reacted with an aminooxy-containing PEG derivative of the form:R-PEG(N)—O—(CH₂)_(n)—O—NH₂where R is methyl, n is 3 and N is approximately 5,000 MW. The purifiedABP containing p-acetylphenylalanine dissolved at 10 mg/mL in 25 mM MES(Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, Mo.) pH 4.5, is reacted with a 10 to 100-fold excess ofaminooxy-containing PEG, and then stirred for 10-16 hours at roomtemperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). The PEG-ABPis then diluted into appropriate buffer for immediate purification andanalysis.

Example 4

Conjugation with a PEG consisting of a hydroxylamine group linked to thePEG via an amide linkage.

A PEG reagent having the following structure is coupled to aketone-containing non-naturally encoded amino acid using the proceduredescribed in Example 3:R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—O—NH₂where R=methyl, n=4 and N is approximately 20,000 MW. The reaction,purification, and analysis conditions are as described in Example 3.

Example 5

This example details the introduction of two distinct non-naturallyencoded amino acids into ABP.

This example demonstrates a method for the generation of anantigen-binding polypeptide that incorporates non-naturally encodedamino acid comprising a ketone functionality at two positions identifiedaccording to Example 1, wherein X* represents a non-naturally encodedamino acid. The antigen-binding polypeptide is prepared as described inExamples 1 and 2, except that the suppressor codon is introduced at twodistinct sites within the nucleic acid.

Example 6

This example details conjugation of antigen-binding polypeptide to ahydrazide-containing PEG and subsequent in situ reduction.

An antigen-binding polypeptide incorporating a carbonyl-containing aminoacid is prepared according to the procedure described in Examples 2 and3. Once modified, a hydrazide-containing PEG having the followingstructure is conjugated to the ABP:R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—X—NH—NH₂where R=methyl, n=2 and N=10,000 MW and X is a carbonyl (C═O) group. Thepurified ABP containing p-acetylphenylalanine is dissolved at between0.1-10 mg/mL in 25 mM MES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mMHepes (Sigma Chemical, St. Louis, Mo.) pH 7.0, or in 10 mM SodiumAcetate (Sigma Chemical, St. Louis, Mo.) pH 4.5, is reacted with a 1 to100-fold excess of hydrazide-containing PEG, and the correspondinghydrazone is reduced in situ by addition of stock 1M NaCNBH₃ (SigmaChemical, St. Louis, Mo.), dissolved in H₂O, to a final concentration of10-50 mM. Reactions are carried out in the dark at 4° C. to RT for 18-24hours. Reactions are stopped by addition of 1 M Tris (Sigma Chemical,St. Louis, Mo.) at about pH 7.6 to a final Tris concentration of 50 mMor diluted into appropriate buffer for immediate purification.

Example 7

This example details introduction of an alkyne-containing amino acidinto an ABP and derivatization with mPEG-azide.

Any of the residues identified according to Example 1 are substitutedwith the following non-naturally encoded amino acid:

The sequences utilized for site-specific incorporation ofp-propargyl-tyrosine are SEQ ID NO: 1 (muttRNA, M. jannaschiimtRNA_(CUA) ^(Tyr)), and 6, 7 or 8 described in Example 2 above. Theantigen-binding polypeptide containing the propargyl tyrosine isexpressed in E. coli and purified using the conditions described inExample 3.

The purified ABP containing propargyl-tyrosine dissolved at between0.1-10 mg/mL in PB buffer (100 mM sodium phosphate, 0.15 M NaCl, pH=8)and a 10 to 1000-fold excess of an azide-containing PEG is added to thereaction mixture. A catalytic amount of CuSO₄ and Cu wire are then addedto the reaction mixture. After the mixture is incubated (including butnot limited to, about 4 hours at room temperature or 37° C., orovernight at 4° C.), H₂O is added and the mixture is filtered through adialysis membrane. The sample can be analyzed for the addition,including but not limited to, by similar procedures described in Example3.

In this Example, the PEG will have the following structure:R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—N₃where R is methyl, n is 4 and N is 10,000 MW.

Example 8

This example details substitution of a large, hydrophobic amino acid inABP with propargyl tyrosine.

A Phe, Trp or Tyr residue present within the sequence of ABP issubstituted with the following non-naturally encoded amino acid asdescribed in Example 7:

Once modified, a PEG is attached to the ABP variant comprising thealkyne-containing amino acid. The PEG will have the following structure:Me-PEG(N)—O—(CH₂)₂—N₃and coupling procedures would follow those in Example 7. This willgenerate a ABP variant comprising a non-naturally encoded amino acidthat is approximately isosteric with one of the naturally-occurring,large hydrophobic amino acids and which is modified with a PEGderivative at a distinct site within the polypeptide.

Example 9

This example details generation of an ABP homodimer, heterodimer,homomultimer, or heteromultimer separated by one or more PEG linkers.

The alkyne-containing ABP variant produced in Example 7 is reacted witha bifunctional PEG derivative of the form:N₃—(CH₂)_(n)—C(O)—NH—(CH₂)₂—O-PEG(N)—O—(CH₂)₂—NH—C(O)—(CH₂)_(n)—N₃where n is 4 and the PEG has an average MW of approximately 5,000, togenerate the corresponding ABP homodimer where the two ABP molecules arephysically separated by PEG. In an analogous manner an antigen-bindingpolypeptide may be coupled to one or more other polypeptides to formheterodimers, homomultimers, or heteromultimers. Coupling, purification,and analyses will be performed as in Examples 7 and 3.

Example 10

This example details coupling of a saccharide moiety to ABP.

One or more amino acid residues of the ABP is substituted with thenon-naturally encoded amino acid below, as described in Example 3.

Once modified, the ABP variant comprising the carbonyl-containing aminoacid is reacted with a β-linked aminooxy analogue of N-acetylglucosamine(GlcNAc). The ABP variant (10 mg/mL) and the aminooxy saccharide (21 mM)are mixed in aqueous 100 mM sodium acetate buffer (pH 5.5) and incubatedat 37° C. for 7 to 26 hours. A second saccharide is coupled to the firstenzymatically by incubating the saccharide-conjugated ABP (5 mg/mL) withUDP-galactose (16 mM) and β-1,4-galactosyltransferase (0.4 units/mL) in150 mM HEPES buffer (pH 7.4) for 48 hours at ambient temperature(Schanbacher et al. J. Biol. Chem. 1970, 245, 5057-5061).

Example 11

This example details generation of a PEGylated ABP antagonist.

One or more of the ABP amino acid residues is substituted with thefollowing non-naturally encoded amino acid as described in Example 3.

Once modified, the ABP variant comprising the carbonyl-containing aminoacid will be reacted with an aminooxy-containing PEG derivative of theform:R-PEG(N)—O—(CH₂)_(n)—O—NH₂where R is methyl, n is 4 and N is 20,000 MW to generate a ABPantagonist comprising a non-naturally encoded amino acid that ismodified with a PEG derivative at a single site within the polypeptide.Coupling, purification, and analyses are performed as in Example 3.

Example 12

Generation of an ABP homodimer, heterodimer, homomultimer, orheteromultimer in which the ABP Molecules are Linked Directly

An ABP variant comprising the alkyne-containing amino acid can bedirectly coupled to another ABP variant comprising the azido-containingamino acid, each of which comprise non-naturally encoded amino acidsubstitutions at the sites described in, but not limited to, Example 10.This will generate the corresponding ABP homodimer where the two ABPvariants are physically joined. In an analogous manner anantigen-binding polypeptide may be coupled to one or more otherpolypeptides to form heterodimers, homomultimers, or heteromultimers.Coupling, purification, and analyses are performed as in Examples 3, 6,and 7.

Example 13

PEG-OH+Br—(CH₂)_(n)—C≡CR′→PEG-O—(CH₂)_(n)—C≡CR′A B

The polyalkylene glycol (P—OH) is reacted with the alkyl halide (A) toform the ether (B). In these compounds, n is an integer from one to nineand R′ can be a straight- or branched-chain, saturated or unsaturatedCl, to C20 alkyl or heteroalkyl group. R′ can also be a C3 to C7saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, asubstituted or unsubstituted aryl or heteroaryl group, or a substitutedor unsubstituted alkaryl (the alkyl is a C1 to C20 saturated orunsaturated alkyl) or heteroalkaryl group. Typically, PEG-OH ispolyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG)having a molecular weight of 800 to 40,000 Daltons (Da).

Example 14

mPEG-OH+Br—CH₂—C≡CH→mPEG-O—CH₂—C≡CH

mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g, 0.1mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL). Asolution of propargyl bromide, dissolved as an 80% weight solution inxylene (0.56 mL, 5 mmol, 50 equiv., Aldrich), and a catalytic amount ofKI were then added to the solution and the resulting mixture was heatedto reflux for 2 hours. Water (1 mL) was then added and the solvent wasremoved under vacuum. To the residue was added CH₂Cl₂ (25 mL) and theorganic layer was separated, dried over anhydrous Na₂SO₄, and the volumewas reduced to approximately 2 mL. This CH₂Cl₂ solution was added todiethyl ether (150 mL) drop-wise. The resulting precipitate wascollected, washed with several portions of cold diethyl ether, and driedto afford propargyl-O-PEG.

Example 15

mPEG-OH+Br—(CH₂)₃—C≡CH→mPEG-O—(CH₂)₃—C≡CH

The mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g,0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL).Fifty equivalents of 5-bromo-1-pentyne (0.53 mL, 5 mmol, Aldrich) and acatalytic amount of KI were then added to the mixture. The resultingmixture was heated to reflux for 16 hours. Water (1 mL) was then addedand the solvent was removed under vacuum. To the residue was addedCH₂Cl₂ (25 mL) and the organic layer was separated, dried over anhydrousNa₂SO₄, and the volume was reduced to approximately 2 mL. This CH₂Cl₂solution was added to diethyl ether (150 mL) drop-wise. The resultingprecipitate was collected, washed with several portions of cold diethylether, and dried to afford the corresponding alkyne. 5-chloro-1-pentynemay be used in a similar reaction.

Example 16

m-HOCH₂C₆H₄OH+NaOH+Br—CH₂—C≡CH→m-HOCH₂C₆H₄O—CH₂—C≡CH  (1)m-HOCH₂C₆H₄O—CH₂—C≡CH+MsCl+N(Et)₃ →m-MsOCH₂C₆H₄O—CH₂—C≡CH  (2)m-MsOCH₂C₆H₄O—CH₂—C≡CH+LiBr→m-Br—CH₂C₆H₄O—CH₂—C≡CH  (3)mPEG-OH+m-Br—CH₂C₆H₄O—CH₂—C≡CH→mPEG-O—CH₂—C₆H₄O—CH₂—C≡CH  (4)

To a solution of 3-hydroxybenzylalcohol (2.4 g, 20 mmol) in THF (50 mL)and water (2.5 mL) was first added powdered sodium hydroxide (1.5 g,37.5 mmol) and then a solution of propargyl bromide, dissolved as an 80%weight solution in xylene (3.36 mL, 30 mmol). The reaction mixture washeated at reflux for 6 hours. To the mixture was added 10% citric acid(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over MgSO₄ andconcentrated to give the 3-propargyloxybenzyl alcohol.

Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of compound 3 (2.0 g, 11.0 mmol) inCH₂Cl₂ at 0° C. and the reaction was placed in the refrigerator for 16hours. A usual work-up afforded the mesylate as a pale yellow oil. Thisoil (2.4 g, 9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g, 23.0mmol) was added. The reaction mixture was heated to reflux for 1 hourand was then cooled to room temperature. To the mixture was added water(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa₂SO₄, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (1.0 g, 0.05 mmol, Sunbio) was dissolved in THF (20 mL)and the solution was cooled in an ice bath. NaH (6 mg, 0.25 mmol) wasadded with vigorous stirring over a period of several minutes followedby addition of the bromide obtained from above (2.55 g, 11.4 mmol) and acatalytic amount of KI. The cooling bath was removed and the resultingmixture was heated to reflux for 12 hours. Water (1.0 mL) was added tothe mixture and the solvent was removed under vacuum. To the residue wasadded CH₂Cl₂ (25 mL) and the organic layer was separated, dried overanhydrous Na₂SO₄, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in a whiteprecipitate, which was collected to yield the PEG derivative.

Example 17

mPEG-NH₂+X—C(O)—(CH₂)_(n)—C≡CR′→mPEG-NH—C(O)—(CH₂)_(n)—C≡CR′

The terminal alkyne-containing poly(ethylene glycol) polymers can alsobe obtained by coupling a poly(ethylene glycol) polymer containing aterminal functional group to a reactive molecule containing the alkynefunctionality as shown above. n is between 1 and 10. R′ can be H or asmall alkyl group from C1 to C4.

Example 18  HO₂C—(CH₂)₂—C≡CH+NHS+DCC→NHSO—C(O)—(CH₂)₂—C≡CH  (1)

mPEG-NH₂+NHSO—C(O)—(CH₂)₂—C≡CH→mPEG-NH—C(O)—(CH₂)₂—C≡CH  (2)

4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH₂Cl₂ (25 mL).N-hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) wereadded and the solution was stirred overnight at room temperature. Theresulting crude NHS ester 7 was used in the following reaction withoutfurther purification.

mPEG-NH₂ with a molecular weight of 5,000 Da (mPEG-NH₂, 1 g, Sunbio) wasdissolved in THF (50 mL) and the mixture was cooled to 4° C. NHS ester 7(400 mg, 0.4 mmol) was added portion-wise with vigorous stirring. Themixture was allowed to stir for 3 hours while warming to roomtemperature. Water (2 mL) was then added and the solvent was removedunder vacuum. To the residue was added CH₂Cl₂ (50 mL) and the organiclayer was separated, dried over anhydrous Na₂SO₄, and the volume wasreduced to approximately 2 mL. This CH₂Cl₂ solution was added to ether(150 mL) drop-wise. The resulting precipitate was collected and dried invacuo.

Example 19

This Example represents the preparation of the methane sulfonyl ester ofpoly(ethylene glycol), which can also be referred to as themethanesulfonate or mesylate of poly(ethylene glycol). The correspondingtosylate and the halides can be prepared by similar procedures.mPEG-OH+CH₃SO₂Cl+N(Et)₃ →mPEG-O—SO₂CH₃ →mPEG-N₃

The mPEG-OH (MW=3,400, 25 g, 10 mmol) in 150 mL of toluene wasazeotropically distilled for 2 hours under nitrogen and the solution wascooled to room temperature. 40 mL of dry CH₂Cl₂ and 2.1 mL of drytriethylamine (15 mmol) were added to the solution. The solution wascooled in an ice bath and 1.2 mL of distilled methanesulfonyl chloride(15 mmol) was added dropwise. The solution was stirred at roomtemperature under nitrogen overnight, and the reaction was quenched byadding 2 mL of absolute ethanol. The mixture was evaporated under vacuumto remove solvents, primarily those other than toluene, filtered,concentrated again under vacuum, and then precipitated into 100 mL ofdiethyl ether. The filtrate was washed with several portions of colddiethyl ether and dried in vacuo to afford the mesylate.

The mesylate (20 g, 8 mmol) was dissolved in 75 ml of THF and thesolution was cooled to 4° C. To the cooled solution was added sodiumazide (1.56 g, 24 mmol). The reaction was heated to reflux undernitrogen for 2 hours. The solvents were then evaporated and the residuediluted with CH₂Cl₂ (50 mL). The organic fraction was washed with NaClsolution and dried over anhydrous MgSO₄. The volume was reduced to 20 mland the product was precipitated by addition to 150 ml of cold dryether.

Example 20

N₃—C₆H₄—CO₂H→N₃—C₆H₄CH₂OH  (1)N₃—C₆H₄CH₂OH→Br—CH₂—C₆H₄—N₃  (2)mPEG-OH+Br—CH₂—C₆H₄—N₃ →mPEG-O—CH₂—C₆H₄—N₃  (3)

4-azidobenzyl alcohol can be produced using the method described in U.S.Pat. No. 5,998,595, which is incorporated by reference herein.Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of 4-azidobenzyl alcohol (1.75 g, 11.0mmol) in CH₂Cl₂ at 0° C. and the reaction was placed in the refrigeratorfor 16 hours. A usual work-up afforded the mesylate as a pale yellowoil. This oil (9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g,23.0 mmol) was added. The reaction mixture was heated to reflux for 1hour and was then cooled to room temperature. To the mixture was addedwater (2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa₂SO₄, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg,0.5 mmol) in THF (35 mL) and the bromide (3.32 g, 15 mmol) was added tothe mixture along with a catalytic amount of KI. The resulting mixturewas heated to reflux for 12 hours. Water (1.0 mL) was added to themixture and the solvent was removed under vacuum. To the residue wasadded CH₂Cl₂ (25 mL) and the organic layer was separated, dried overanhydrous Na₂SO₄, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in aprecipitate, which was collected to yield mPEG-O—CH₂—C₆H₄—N₃.

Example 21

NH₂—PEG-O—CH₂CH₂CO₂H+N₃—CH₂CH₂CO₂—NHS→N₃—CH₂CH₂—C(O)NH-PEG-O—CH₂CH₂CO₂H

NH₂—PEG-O—CH₂CH₂CO₂H (MW 3,400 Da, 2.0 g) was dissolved in a saturatedaqueous solution of NaHCO₃ (10 mL) and the solution was cooled to 0° C.3-azido-1-N-hydroxysuccinimido propionate (5 equiv.) was added withvigorous stirring. After 3 hours, 20 mL of H₂O was added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 NH₂SO₄ and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH₂Cl₂(100 mL×3), dried over Na₂SO₄ and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the omega-carboxy-azide PEG derivative.

Example 22

mPEG-OMs+HC≡CLi→mPEG-O—CH₂—CH₂—C≡C—H

To a solution of lithium acetylide (4 equiv.), prepared as known in theart and cooled to −78° C. in THF, is added dropwise a solution ofmPEG-OMs dissolved in THF with vigorous stirring. After 3 hours, thereaction is permitted to warm to room temperature and quenched with theaddition of 1 mL of butanol. 20 mL of H₂O is then added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 NH₂SO₄ and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH₂Cl₂(100 mL×3), dried over Na₂SO₄ and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the 1-(but-3-ynyloxy)-methoxypolyethylene glycol(mPEG).

Example 23

The azide- and acetylene-containing amino acids were incorporatedsite-selectively into proteins using the methods described in L. Wang,et al., (2001), Science 292:498-500, J. W. Chin et al., Science301:964-7 (2003)), J. W. Chin et al., (2002), Journal of the AmericanChemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002),Chem Bio Chem 11:1135-1137; J. W. Chin, et al., (2002), PNAS UnitedStates of America 99:11020-11024: and, L. Wang, & P. G. Schultz, (2002),Chem. Comm., 1-10. Once the amino acids were incorporated, thecycloaddition reaction was carried out with 0.01 mM protein in phosphatebuffer (PB), pH 8, in the presence of 2 mM PEG derivative, 1 mM CuSO₄,and ˜1 mg Cu-wire for 4 hours at 37° C.

Example 24

This example describes methods to measure in vitro and in vivo activityof ABP comprising a non-naturally encoded amino acid and PEGylated ABP.

Cell Binding Assays

Cells (3×10⁶) are incubated in duplicate in PBS/1% BSA (100 μl) in theabsence or presence of various concentrations (volume: 10 μl) ofunlabeled ABP, ABP or a negative control and in the presence of ¹²⁵I-ABP(approx. 100,000 cpm or 1 ng) at 0° C. for 90 minutes (total volume: 120μl). Cells are then resuspended and layered over 200 μl ice cold FCS ina 350 μl plastic centrifuge tube and centrifuged (1000 g; 1 minute). Thepellet is collected by cutting off the end of the tube and pellet andsupernatant counted separately in a gamma counter (Packard).

Specific binding (cpm) is determined as total binding in the absence ofa competitor (mean of duplicates) minus binding (cpm) in the presence of100-fold excess of unlabeled ABP (non-specific binding). Thenon-specific binding is measured for each of the cell types used.Experiments are run on separate days using the same preparation of¹²⁵I-ABP and should display internal consistency. The binding isinhibited in a dose dependent manner by unlabeled natural ABP or ABP,but not by the negative control. The ability of ABP to compete for thebinding of natural ¹²⁵I-ABP suggests that the receptors recognize bothforms equally well.

For binding assays with scFv-108, A431 cells were collected followingtreatment with trypsin, re-suspended in FACS buffer (PBS, 2% FBS, 0.01%NaN₃), and then seeded into 96-well round bottom microtiter plates(3×10⁵ cell/well). Cells were incubated with different concentrations ofwild type or pAcF-containing scFv-108 fragments for 30 minutes on ice.Unbound scFv proteins were removed by washing following centrifugation(repeated 2-3 times). Cells were then incubated with the mAb-108 (ATCC #HB 9764) at a concentration of 7.5 nM (EC₈₀) for 30 minutes. After twowashes, the cells were incubated with an APC-labeled (allophycocyanin)anti-mouse antibody (10 ug/ml) for 30 minutes on ice. After washing thecells two times to remove the secondary antibody, the cells werere-suspended in FACS buffer supplemented with propidium iodine (0.5ug/ml), and analyzed by flow cytometry. For binding assays with Fab-108,cells were incubated with increasing amounts of Fab-108 under the sameconditions as used for the scFv-108 assays. Fab binding was detectedusing an anti-His antibody followed by an APC-labeled anti-mousesecondary antibody.

FIG. 7, Panels A-C shows competition binding curves of the scFv proteinscontaining p-acetyl-phenylalanine (pAcF) or pAcF with PEG and WT scFv toA431 cells expressing EGF receptors. Cells were incubated with the scFvproteins at various concentrations after washing to remove unboundscFv's, and the cells were treated with the mAb108 as described above.All proteins were expressed in the periplasm. Table 11 summarizes thebinding of the modified scFv's (pAcF and pAcF with PEG) relative to thewild type scFv: TABLE 11 scFv 108 IC50 WT 1x (8.1 nm) Ser131pAcF 1.3xSer131pAcF-5K PEG 5.0x Ser136pAcF 1.6x Ser136pAcF-5K PEG 6.3x His144pAcF2.6x Leu156pAcF 1.8x Leu156pAcF-5K PEG 2.9x Tyr190pAcF 2.1x Ser193pAcF1.8x Lys248pAcF 2.1x Ser259pAcF 0.5x

FIG. 8, Panels B-D show binding of pAcF or pAcF-PEG-containing Fab-108fragments and WT Fab to A431 cells expressing EGF receptors. PEGylationof the Fab fragments results in a minimal decrease in the affinity ofthe fragments to the EGF receptors. Binding of modified Fab fragmentsrelative to that of the wild type Fab is shown in Table 12. Bindingconditions were as described previously. TABLE 12 Fab108 EC50 WT 1x (4.0nM) Lys142pAcF 1.7x Lys142pAcF- 2.8x 5K PEG Thr204pAcF 1.7x Thr204pAcF-1.8x 5K PEG Lys 219pAcF 2.0x

In Vivo Studies of PEGylated ABP

PEG-ABP, unmodified ABP and buffer solution are administered to mice orrats. The results will show superior activity and prolonged half life ofthe PEGylated ABP of the present invention compared to unmodified ABP.

Measurement of the In Vivo Half-Life of Conjugated and Non-ConjugatedABP and Variants Thereof.

Male Sprague Dawley rats (about 7 weeks old) are used. On the day ofadministration, the weight of each animal is measured. 100 μg per kgbody weight of the non-conjugated and conjugated ABP samples are eachinjected intravenously into the tail vein of three rats. At 1 minute, 30minutes, 1, 2, 4, 6, and 24 hours after the injection, 500 μl of bloodis withdrawn from each rat while under CO₂-anesthesia. The blood samplesare stored at room temperature for 1.5 hours followed by isolation ofserum by centrifugation (4° C., 18000×g for 5 minutes). The serumsamples are stored at −80° C. until the day of analysis. The amount ofactive ABP in the serum samples is quantified by the ABP in vitroactivity assay after thawing the samples on ice.

Example 31

Human Clinical Trial of the Safety and/or Efficacy of PEGylated ABPComprising a Non-Naturally Encoded Amino Acid.

Objective To compare the safety and pharmacokinetics of subcutaneouslyadministered PEGylated recombinant human ABP comprising a non-naturallyencoded amino acid with a commercially available product specific forthe same target antigen (e.g. Herceptin®, Bexxar®, Campath®, CEA-Scan®,Enbrel®, Erbitux®, Humira®, Myoscint®, Prostascint®, Raptiva®,Remicade®, ReoPro®, Rituxan®, Simulect®, Synagis®, Verluma®, Xolair®,Zenapax®, Zevalin®, or Avastin®.

Patients Eighteen healthy volunteers ranging between 20-40 years of ageand weighing between 60-90 kg are enrolled in the study. The subjectswill have no clinically significant abnormal laboratory values forhematology or serum chemistry, and a negative urine toxicology screen,HIV screen, and hepatitis B surface antigen. They should not have anyevidence of the following: hypertension; a history of any primaryhematologic disease; history of significant hepatic, renal,cardiovascular, gastrointestinal, genitourinary, metabolic, neurologicdisease; a history of anemia or seizure disorder; a known sensitivity tobacterial or mammalian-derived products, PEG, or human serum albumin;habitual and heavy consumer to beverages containing caffeine;participation in any other clinical trial or had blood transfused ordonated within 30 days of study entry; had exposure to ABP within threemonths of study entry; had an illness within seven days of study entry;and have significant abnormalities on the pre-study physical examinationor the clinical laboratory evaluations within 14 days of study entry.All subjects are evaluable for safety and all blood collections forpharmacokinetic analysis are collected as scheduled. All studies areperformed with institutional ethics committee approval and patientconsent.

Study Design This will be a Phase I, single-center, open-label,randomized, two-period crossover study in healthy male volunteers.Eighteen subjects are randomly assigned to one of two treatment sequencegroups (nine subjects/group). ABP is administered over two separatedosing periods as a bolus s.c. injection in the upper thigh usingequivalent doses of the PEGylated ABP comprising a non-naturally encodedamino acid and the commercially available product chosen. The dose andfrequency of administration of the commercially available product is asinstructed in the package label. Additional dosing, dosing frequency, orother parameter as desired, using the commercially available productsmay be added to the study by including additional groups of subjects.Each dosing period is separated by a 14-day washout period. Subjects areconfined to the study center at least 12 hours prior to and 72 hoursfollowing dosing for each of the two dosing periods, but not betweendosing periods. Additional groups of subjects may be added if there areto be additional dosing, frequency, or other parameter, to be tested forthe PEGylated ABP as well. The experimental formulation of ABP is thePEGylated ABP comprising a non-naturally encoded amino acid.

Blood Sampling Serial blood is drawn by direct vein puncture before andafter administration of ABP. Venous blood samples (5 mL) fordetermination of serum ABP concentrations are obtained at about 30, 20,and 10 minutes prior to dosing (3 baseline samples) and at approximatelythe following times after dosing: 30 minutes and at 1, 2, 5, 8, 12, 15,18, 24, 30, 36, 48, 60 and 72 hours. Each serum sample is divided intotwo aliquots. All serum samples are stored at −20° C. Serum samples areshipped on dry ice. Fasting clinical laboratory tests (hematology, serumchemistry, and urinalysis) are performed immediately prior to theinitial dose on day 1, the morning of day 4, immediately prior to dosingon day 16, and the morning of day 19.

Bioanalytical Methods A radioimmunoassay (RA) or ELISA kit procedure isused for the determination of serum ABP concentrations.

Safety Determinations Vital signs are recorded immediately prior to eachdosing (Days 1 and 16), and at 6, 24, 48, and 72 hours after eachdosing. Safety determinations are based on the incidence and type ofadverse events and the changes in clinical laboratory tests frombaseline. In addition, changes from pre-study in vital signmeasurements, including blood pressure, and physical examination resultsare evaluated.

Data Analysis Post-dose serum concentration values are corrected forpre-dose baseline ABP concentrations by subtracting from each of thepost-dose values the mean baseline ABP concentration determined fromaveraging the ABP levels from the three samples collected at 30, 20, and10 minutes before dosing. Pre-dose serum ABP concentrations are notincluded in the calculation of the mean value if they are below thequantification level of the assay. Pharmacokinetic parameters aredetermined from serum concentration data corrected for baseline ABPconcentrations. Pharmacokinetic parameters are calculated by modelindependent methods on a Digital Equipment Corporation VAX 8600 computersystem using the latest version of the BIOAVL software. The followingpharmacokinetics parameters are determined: peak serum concentration(C_(max)); time to peak serum concentration (t_(max)); area under theconcentration-time curve (AUC) from time zero to the last blood samplingtime (AUC₀₋₇₂) calculated with the use of the linear trapezoidal rule;and terminal elimination half-life (t_(1/2)), computed from theelimination rate constant. The elimination rate constant is estimated bylinear regression of consecutive data points in the terminal linearregion of the log-linear concentration-time plot. The mean, standarddeviation (SD), and coefficient of variation (CV) of the pharmacokineticparameters are calculated for each treatment. The ratio of the parametermeans (preserved formulation/non-preserved formulation) is calculated.

Safety Results The incidence of adverse events is equally distributedacross the treatment groups. There are no clinically significant changesfrom baseline or pre-study clinical laboratory tests or blood pressures,and no notable changes from pre-study in physical examination resultsand vital sign measurements. The safety profiles for the two treatmentgroups should appear similar.

Pharmacokinetic Results Mean serum ABP concentration-time profiles(uncorrected for baseline ABP levels) in all 18 subjects after receivinga single dose of one or more of commercially available products specificfor the same target antigen are compared to the PEGylated ABP comprisinga non-naturally encoded amino acid at each time point measured. Allsubjects should have pre-dose baseline ABP concentrations within thenormal physiologic range. Pharmacokinetic parameters are determined fromserum data corrected for pre-dose mean baseline ABP concentrations andthe C_(max) and t_(max) are determined. The mean t_(max) for theclinical comparator(s) chosen is significantly shorter than the t_(max)for the PEGylated ABP comprising the non-naturally encoded amino acid.Terminal half-life values are significantly shorter for the commerciallyavailable ABP products tested compared with the terminal half-life forthe PEGylated ABP comprising a non-naturally encoded amino acid.

Although the present study is conducted in healthy male subjects,similar absorption characteristics and safety profiles would beanticipated in other patient populations; such as male or femalepatients with cancer or chronic renal failure, pediatric renal failurepatients, patients in autologous predeposit programs, or patientsscheduled for elective surgery.

In conclusion, subcutaneously administered single doses of PEGylated ABPcomprising non-naturally encoded amino acid will be safe and welltolerated by healthy male subjects. Based on a comparative incidence ofadverse events, clinical laboratory values, vital signs, and physicalexamination results, the safety profiles of the commercially availableforms of ABP and PEGylated ABP comprising non-naturally encoded aminoacid will be equivalent. The PEGylated ABP comprising non-naturallyencoded amino acid potentially provides large clinical utility topatients and health care providers.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference herein intheir entirety for all purposes.

ADDITIONAL AND ALTERNATE EMBODIMENTS OF THE INVENTION

1. An antigen-binding polypeptide comprising one or more non-naturallyencoded amino acids.

2. The antigen-binding polypeptide of claim 1, wherein theantigen-binding polypeptide comprises one or more post-translationalmodifications.

3. The antigen-binding polypeptide of claim 1, wherein theantigen-binding polypeptide is linked to a linker, polymer, orbiologically active molecule.

4. The antigen-binding polypeptide of claim 3, wherein the polypeptideis linked to a water soluble polymer.

5. The antigen-binding polypeptide of claim 1, wherein the polypeptideis linked to a bifunctional polymer, bifunctional linker, or at leastone additional antigen-binding polypeptide.

6. The antigen-binding polypeptide of claim 5, wherein the bifunctionallinker or polymer is linked to a second polypeptide.

7. The antigen-binding polypeptide of claim 6, wherein the secondpolypeptide is an antigen-binding polypeptide.

8. The antigen-binding polypeptide of claim 6, wherein the secondpolypeptide is a non-antigen-binding polypeptide.

9. The antigen-binding polypeptide of claim 4, wherein the water solublepolymer comprises a poly(ethylene glycol) moiety.

10. The antigen-binding polypeptide of claim 5, wherein the bifunctionalpolymer is a poly(ethylene glycol) moiety.

11. The antigen-binding polypeptide of claim 4, wherein said watersoluble polymer is linked to a non-naturally encoded amino acid presentin said antigen-binding polypeptide.

12. The antigen-binding polypeptide of claim 1, comprising at least twoamino acids linked to a water soluble polymer comprising a poly(ethyleneglycol) moiety.

13. The antigen-binding polypeptide of claim 12, wherein at least oneamino acid linked to said water soluble polymer is a non-naturallyencoded amino acid.

14. The antigen-binding polypeptide of claim 1, wherein theantigen-binding polypeptide comprises one or more amino acidsubstitution, addition or deletion that modulates affinity of theantigen-binding polypeptide for an ABP receptor or antigen.

15. The antigen-binding polypeptide of claim 1, wherein theantigen-binding polypeptide comprises one or more amino acidsubstitution, addition or deletion that modulates the stability,expression level in a recombinant host cell or synthesized in vitro,immunogenicity, protease resistance, tissue or organ specificity, orsolubility of the antigen-binding polypeptide.

16. The antigen-binding polypeptide of claim 1, wherein thenon-naturally encoded amino acid is reactive toward a linker, polymer,or biologically active molecule that is otherwise unreactive toward anyof the 20 common amino acids in the polypeptide.

17. The antigen-binding polypeptide of claim 1, wherein thenon-naturally encoded amino acid comprises a carbonyl group, an aminooxygroup, a hydrazine group, a hydrazide group, a semicarbazide group, anazide group, or an alkyne group.

18. The antigen-binding polypeptide of claim 17, wherein thenon-naturally encoded amino acid comprises a carbonyl group.

19. The antigen-binding polypeptide of claim 18, wherein thenon-naturally encoded amino acid has the structure:

wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R2 is H, an alkyl, aryl, substituted alkyl, andsubstituted aryl; and R3 is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R4 is H, an amino acid, a polypeptide,or a carboxy terminus modification group.20. The antigen-binding polypeptide of claim 17, wherein thenon-naturally encoded amino acid comprises an aminooxy group.21. The antigen-binding polypeptide of claim 17, wherein thenon-naturally encoded amino acid comprises a hydrazide group.22. The antigen-binding polypeptide of claim 17, wherein thenon-naturally encoded amino acid comprises a hydrazine group.23. The antigen-binding polypeptide of claim 17, wherein thenon-naturally encoded amino acid comprises a semicarbazide group.24. The antigen-binding polypeptide of claim 17, wherein thenon-naturally encoded amino acid comprises an azide group.25. The antigen-binding polypeptide of claim 24, wherein thenon-naturally encoded amino acid has the structure:

wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R2 is H, anamino acid, a polypeptide, or an amino terminus modification group, andR3 is H, an amino acid, pa polypeptide, or a carboxy terminusmodification group.26. The antigen-binding polypeptide of claim 17, wherein thenon-naturally encoded amino acid comprises an alkyne group.27. The antigen-binding polypeptide of claim 26, wherein thenon-naturally encoded amino acid has the structure:

wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, orsubstituted aryl; X is O, N, S or not present; m is 0-10, R2 is H, anamino acid, a polypeptide, or an amino terminus modification group, andR3 is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.28. The antigen-binding polypeptide of claim 4, wherein the watersoluble polymer has a molecular weight of between about 0.1 kDa andabout 100 kDa.29. The antigen-binding polypeptide of claim 28, wherein the watersoluble polymer has a molecular weight of between about 0.1 kDa andabout 50 kDa.30. The antigen-binding polypeptide of claim 4, which is made byreacting an antigen-binding polypeptide comprising a carbonyl-containingamino acid with a water soluble polymer comprising an aminooxy, ahydrazine, hydrazide or semicarbazide group.31. The antigen-binding polypeptide of claim 30, wherein the aminooxy,hydrazine, hydrazide or semicarbazide group is linked to the watersoluble polymer through an amide linkage.32. The antigen-binding polypeptide of claim 4, which is made byreacting a water soluble polymer comprising a carbonyl group with apolypeptide comprising a non-naturally encoded amino acid that comprisesan aminooxy, a hydrazine, a hydrazide or a semicarbazide group.33. The antigen-binding polypeptide of claim 4, which is made byreacting an antigen-binding polypeptide comprising an alkyne-containingamino acid with a water soluble polymer comprising an azide moiety.34. The antigen-binding polypeptide of claim 4, which is made byreacting an antigen-binding polypeptide comprising an azide-containingamino acid with a water soluble polymer comprising an alkyne moiety.35. The antigen-binding polypeptide of claim 17, wherein the azide oralkyne group is linked to a water soluble polymer through an amidelinkage.36. The antigen-binding polypeptide of claim 4, wherein the watersoluble polymer is a branched or multiarmed polymer.37. The antigen-binding polypeptide of claim 36, wherein each branch ofthe water soluble polymer has a molecular weight of between about 1 kDaand 100 kDa.38. The antigen-binding polypeptide of claim 1, wherein the polypeptideis an antigen-binding polypeptide antagonist.39. The antigen-binding polypeptide of claim 38, wherein the polypeptidecomprises one or more post-translational modification, linker, polymer,or biologically active molecule.40. The antigen-binding polypeptide of claim 39, wherein the polymercomprises a moiety selected from a group consisting of a water solublepolymer and poly(ethylene glycol).41. The antigen-binding polypeptide of claim 1, wherein thenon-naturally encoded amino acid comprises a saccharide moiety.42. The antigen-binding polypeptide of claim 3, wherein the linker,polymer, or biologically active molecule is linked to the polypeptidevia a saccharide moiety.43. An isolated nucleic acid comprising a polynucleotide that hybridizesunder stringent conditions to an antigen-binding polypeptide-encodingpolynucleotide, wherein the polynucleotide comprises at least oneselector codon.44. The isolated nucleic acid of claim 43, wherein the selector codon isselected from the group consisting of an amber codon, ochre codon, opalcodon, a unique codon, a rare codon, and a four-base codon.45. A method of making the antigen-binding polypeptide of claim 3, themethod comprising contacting an isolated antigen-binding polypeptidecomprising a non-naturally encoded amino acid with a linker, polymer, orbiologically active molecule comprising a moiety that reacts with thenon-naturally encoded amino acid.46. The method of claim 45, wherein the polymer comprises a moietyselected from a group consisting of a water soluble polymer andpoly(ethylene glycol).47. The method of claim 45, wherein the non-naturally encoded amino acidcomprises a carbonyl group, an aminooxy group, a hydrazide group, ahydrazine group, a semicarbazide group, an azide group, or an alkynegroup.48. The method of claim 45, wherein the non-naturally encoded amino acidcomprises a carbonyl moiety and the linker, polymer, or biologicallyactive molecule comprises an aminooxy, a hydrazine, a hydrazide, or asemicarbazide moiety.49. The method of claim 48, wherein the aminooxy, hydrazine, hydrazide,or semicarbazide moiety is linked to the linker, polymer, orbiologically active molecule through an amide linkage.50. The method of claim 45, wherein the non-naturally encoded amino acidcomprises an alkyne moiety and the linker, polymer, or biologicallyactive molecule comprises an azide moiety.51. The method of claim 45, wherein the non-naturally encoded amino acidcomprises an azide moiety and the linker, polymer, or biologicallyactive molecule comprises an alkyne moiety.52. The method of claim 47, wherein the azide or alkyne moiety is linkedto a linker, polymer, or biologically active molecule through an amidelinkage.53. The method of claim 46, wherein the poly(ethylene glycol) moiety hasan average molecular weight of between about 0.1 kDa and about 100 kDa.54. The method of claim 46, wherein the poly(ethylene glycol) moiety isa branched or multiarmed polymer.55. A composition comprising the antigen-binding polypeptide of claim 1and a pharmaceutically acceptable carrier.56. The composition of claim 55, wherein the non-naturally encoded aminoacid is linked to a water soluble polymer.57. A method of treating a patient having a disorder modulated by ABPcomprising administering to the patient a therapeutically-effectiveamount of the composition of claim 55.58. A cell comprising the nucleic acid of claim 43.59. The cell of claim 58, wherein the cell comprises an orthogonal tRNAsynthetase or an orthogonal tRNA.60. A method of making an antigen-binding polypeptide comprising anon-naturally encoded amino acid, the method comprising, culturing cellscomprising a polynucleotide or polynucleotides encoding anantigen-binding polypeptide comprising a selector codon, an orthogonalRNA synthetase and an orthogonal tRNA under conditions to permitexpression of the antigen-binding polypeptide comprising a non-naturallyencoded amino acid; and purifying the antigen-binding polypeptide.61. A method of modulating serum half-life or circulation time of anantigen-binding polypeptide, the method comprising substituting one ormore non-naturally encoded amino acids for any one or more naturallyoccurring amino acids in the antigen-binding polypeptide.62. An antigen-binding polypeptide encoded by a polynucleotide whereinsaid polynucleotide comprises a selector codon, and wherein saidpolypeptide comprises at least one non-naturally encoded amino acid.63. The antigen-binding polypeptide of claim 62, wherein thenon-naturally encoded amino acid is linked to a linker, polymer, watersoluble polymer, or biologically active molecule.64. The antigen-binding polypeptide of claim 63, wherein the watersoluble polymer comprises a poly(ethylene glycol) moiety.65. The antigen-binding polypeptide of claim 62, wherein thenon-naturally encoded amino acid comprises a carbonyl group, an aminooxygroup, a hydrazide group, a hydrazine group, a semicarbazide group, anazide group, or an alkyne group.66. The antigen-binding polypeptide of claim 64, wherein thepoly(ethylene glycol) moiety has a molecular weight of between about 0.1kDa and about 100 kDa.67. The antigen-binding polypeptide of claim 64, wherein thepoly(ethylene glycol) moiety is a branched or multiarmed polymer.68. The antigen-binding polypeptide of claim 67, wherein thepoly(ethylene glycol) moiety has a molecular weight of between about 1kDa and about 100 kDa.69. A composition comprising the antigen-binding polypeptide of claim 62and a pharmaceutically acceptable carrier.70. A bispecific ABP comprising a first ABP and a second ABP joined toeach other wherein said first ABP and said second ABP bind specificallyto different epitopes wherein said first ABP has binding specificity forat least one epitope on a first antigen, and the second ABP has bindingspecificity for a second epitope on the first antigen or a secondantigen which is different from said first epitope, and wherein saidbispecific ABP comprises at least one non-naturally encoded amino acid.71. The bispecific ABP of claim 70, wherein said first ABP and saidsecond ABP are joined by a linker.72. The bispecific ABP of claim 71, wherein said linker is a peptidelinker.73. The bispecific ABP of claim 72, wherein said linker is a peptidelinker that lacks a proteolytic cleavage site.74. The bispecific ABP of claim 70, wherein said first ABP is a singlechain ABP and said second ABP is a single chain ABP and said first ABPis coupled to said second ABP by a peptide linker.75. A composition comprising a bispecific ABP of claim 70 and apharmaceutically acceptable carrier.76. A method for treating a disease or condition, said method comprisingadministering to a patient in need thereof a therapeutically effectiveamount of the composition of claim 75.77. An ABP comprising the bispecific ABP of claim 70, coupled to abiologically active molecule.78. The ABP of claim 77, wherein said biologically active molecule isselected from the group consisting of a cytotoxin, a label, aradionuclide, a drug, a liposome, a ligand, and an ABP.79. The ABP of claim 77, wherein said ABP is a fusion protein.80. A method of detecting a cell or tissue expressing one or moreantigens, said method comprising: contacting a cell or tissue with anABP of claim 70 attached to a detectable label; and detecting said labelwherein detection of said label in association with a cell or tissueindicates the presence of a cell or tissue expressing one or moreantigens bound by the ABP.81. The method of claim 80, wherein said detectable label is selectedfrom the group consisting of a gamma emitter, a positron emitter, an MRIlabel, and a fluorescent label.82. The method of claim 80, wherein said detectable label is a gammaemitter and said detecting comprises imaging with a gamma camera.83. The method of claim 80, wherein said detectable label is a positronemitter and said detecting comprises imaging with positron emissiontomography (PET).84. The method of claim 80, wherein said detectable label is an MRIlabel and said detecting comprises detecting with magnetic resonanceimaging.85. An antigen-binding polypeptide comprising a water soluble polymerlinked by a covalent bond to the antigen-binding polypeptide at a singleamino acid.86. The antigen-binding polypeptide of claim 85, wherein the watersoluble polymer comprises a poly(ethylene glycol) moiety.87. The antigen-binding polypeptide of claim 85, wherein the amino acidcovalently linked to the water soluble polymer is a non-naturallyencoded amino acid.88. An antigen-binding polypeptide comprising at least one linker,polymer, or biologically active molecule, wherein said linker, polymer,or biologically active molecule is attached to the polypeptide through afunctional group of a non-naturally encoded amino acid ribosomallyincorporated into the polypeptide.89. The antigen-binding polypeptide of claim 88, wherein saidantigen-binding polypeptide is monoPEGylated.90. An antigen-binding polypeptide comprising a linker, polymer, orbiologically active molecule that is attached to one or morenon-naturally encoded amino acids wherein said non-naturally encodedamino acid is ribosomally incorporated into the polypeptide atpre-selected sites.91. The antigen-binding polypeptide of claim 90, wherein theantigen-binding polypeptide comprises one said linker, polymer, orbiologically active molecule.92. The antigen-binding polypeptide of claim 1, wherein theantigen-binding polypeptide comprises one or more amino acidsubstitution, addition, or deletion that modulates serum half-life orcirculation time of the antigen-binding polypeptide.93. A method of modulating immunogenicity of an antigen-bindingpolypeptide, the method comprising substituting one or morenon-naturally encoded amino acids for any one or more naturallyoccurring amino acids in the antigen-binding polypeptide.94. The isolated nucleic acid of claim 43, wherein the sequence of theisolated nucleic acid is selected from the group consisting of SEQ IDNO: 18, 20, 22, 25, 27, and 29 or fragment thereof.95. An antigen-binding polypeptide wherein the polypeptide is selectedfrom the group consisting of SEQ ID NO: 19, 21, 23, 24, 26, 28, 30, 31or fragment thereof.96. The antigen-binding polypeptide of claim 3, wherein theantigen-binding polypeptide is linked to the linker, polymer, orbiologically active molecule under denaturing conditions.

1. An antigen-binding polypeptide comprising one or more non-naturallyencoded amino acids.
 2. The antigen-binding polypeptide of claim 1,wherein the antigen-binding polypeptide comprises one or morepost-translational modifications.
 3. The antigen-binding polypeptide ofclaim 1, wherein the antigen-binding polypeptide is linked to a linker,polymer, or biologically active molecule.
 4. An isolated nucleic acidcomprising a polynucleotide that hybridizes under stringent conditionsto an antigen-binding polypeptide-encoding polynucleotide, wherein thepolynucleotide comprises at least one selector codon.
 5. A method ofmaking the antigen-binding polypeptide of claim 3, the method comprisingcontacting an isolated antigen-binding polypeptide comprising anon-naturally encoded amino acid with a linker, polymer, or biologicallyactive molecule comprising a moiety that reacts with the non-naturallyencoded amino acid.
 6. A composition comprising the antigen-bindingpolypeptide of claim 1 and a pharmaceutically acceptable carrier.
 7. Amethod of treating a patient having a disorder modulated by ABPcomprising administering to the patient a therapeutically-effectiveamount of the composition of claim
 6. 8. A cell comprising the nucleicacid of claim
 4. 9. A method of making an antigen-binding polypeptidecomprising a non-naturally encoded amino acid, the method comprising,culturing cells comprising a polynucleotide or polynucleotides encodingan antigen-binding polypeptide comprising a selector codon, anorthogonal RNA synthetase and an orthogonal tRNA under conditions topermit expression of the antigen-binding polypeptide comprising anon-naturally encoded amino acid; and purifying the antigen-bindingpolypeptide.
 10. A method of modulating serum half-life or circulationtime of an antigen-binding polypeptide, the method comprisingsubstituting one or more non-naturally encoded amino acids for any oneor more naturally occurring amino acids in the antigen-bindingpolypeptide.
 11. An antigen-binding polypeptide encoded by apolynucleotide wherein said polynucleotide comprises a selector codon,and wherein said polypeptide comprises at least one non-naturallyencoded amino acid.
 12. A composition comprising the antigen-bindingpolypeptide of claim 11 and a pharmaceutically acceptable carrier.
 13. Abispecific ABP comprising a first ABP and a second ABP joined to eachother wherein said first ABP and said second ABP bind specifically todifferent epitopes wherein said first ABP has binding specificity for atleast one epitope on a first antigen, and the second ABP has bindingspecificity for a second epitope on the first antigen or a secondantigen which is different from said first epitope, and wherein saidbispecific ABP comprises at least one non-naturally encoded amino acid.14. A composition comprising a bispecific ABP of claim 13 and apharmaceutically acceptable carrier.
 15. A method for treating a diseaseor condition, said method comprising administering to a patient in needthereof a therapeutically effective amount of the composition of claim14.
 16. An ABP comprising the bispecific ABP of claim 13, coupled to abiologically active molecule.
 17. A method of detecting a cell or tissueexpressing one or more antigens, said method comprising: contacting acell or tissue with an ABP of claim 13 attached to a detectable label;and detecting said label wherein detection of said label in associationwith a cell or tissue indicates the presence of a cell or tissueexpressing one or more antigens bound by the ABP.
 18. An antigen-bindingpolypeptide comprising a water soluble polymer linked by a covalent bondto the antigen-binding polypeptide at a single amino acid.
 19. Anantigen-binding polypeptide comprising at least one linker, polymer, orbiologically active molecule, wherein said linker, polymer, orbiologically active molecule is attached to the polypeptide through afunctional group of a non-naturally encoded amino acid ribosomallyincorporated into the polypeptide.
 20. An antigen-binding polypeptidecomprising a linker, polymer, or biologically active molecule that isattached to one or more non-naturally encoded amino acids wherein saidnon-naturally encoded amino acid is ribosomally incorporated into thepolypeptide at pre-selected sites.
 21. A method of modulatingimmunogenicity of an antigen-binding polypeptide, the method comprisingsubstituting one or more non-naturally encoded amino acids for any oneor more naturally occurring amino acids in the antigen-bindingpolypeptide.
 22. An antigen-binding polypeptide wherein the polypeptideis selected from the group consisting of SEQ ID NO: 19, 21, 23, 24, 26,28, 30, 31 or fragment thereof.
 23. The antigen-binding polypeptide ofclaim 3, wherein the antigen-binding polypeptide is linked to thelinker, polymer, or biologically active molecule under denaturingconditions.